BE115's Tissue Engineering Blog

Fibrillin-1 and -2 differentially modulate endogenous TGF-β and BMP bioavailability during bone formation

2010-11-01T16:58:00.000-07:00

Harikiran Nistala, Sui Lee-Arteaga, Silvia Smaldone, Gabriella Siciliano, Luca Carta, Robert N. Ono, Gerhard Sengle, Emilio Arteaga-Solis, Regis Levasseur, Patricia Ducy, Lynn Y. Sakai, Gerard Karsenty, and Francesco Ramirez

ABSTRACT

Extracellular regulation of signaling by transforming growth factor (TGF)–β family members is emerging as a key aspect of organ formation and tissue remodeling. In this study, we demonstrate that fibrillin-1 and -2, the structural components of extracellular microfibrils, differentially regulate TGF-β and bone morphogenetic protein (BMP) bioavailability in bone. Fibrillin-2–null (Fbn2-/-) mice display a low bone mass phenotype that is associated with reduced bone formation in vivo and impaired osteoblast maturation in vitro. This Fbn2-/- phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization. Cultured osteoblasts from Fbn1-/- mice exhibit improper latent TGF-β activation as well, but mature faster because of increased availability of otherwise matrix-bound BMPs. Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition. Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

SUMMARY

INTRODUCTION

The extra-cellular matrix (ECM) has been shown to be an important component of growth signaling and development. Amongst the ECM proteins, fibrillins and their associated proteins have been found to be important for cell maturation and growth and are typically bound by TGF-β and BMP. This paper looks at the effects of mutated proteins/genes for fibrillin-1 and -2 individually, focusing on TGF-β and BMP and their effects on osteoblast growth in the context of Marfan Syndrom (MFS) and congenital contractural arachnodactyly (CCA).

Figure 9. Model of microfibril-mediated control of osteoblast maturation. The scheme summarizes the distinct contributions of osteoblast-produced fibrillin-1 and fibrillin-2 microfibrils to osteogenic differentiation through the differential regulation of endogenous TGF-β and BMP signals that together calibrate the rate of bone formation.

RESULTS

Figure 1. Reduced bone mass and BFR in Fbn2-/- mice. (A) Representative von Kossa staining and μCT images of vertebral sections from 3-mo-old WT and Fbn2-/- male mice with histograms summarizing the μCT measurements of volumetric bone mineral density (BMD) and BV/TV in these samples. (B) Illustrative examples of dual-calcein labeling in tibiae of 3-mo-old WT and Fbn2-/- male mice with histograms summarizing BFR values and osteoblast numbers in WT and mutant samples. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < src="http://2.bp.blogspot.com/_GjNXhZPfwZI/TM_K4-VpS4I/AAAAAAAAAAU/AorR8VPPq4o/s200/fig2.bmp" style="display: block; margin: 0px auto 10px; text-align: center; cursor: pointer; width: 178px; height: 200px;" alt="" id="BLOGGER_PHOTO_ID_5534865547216898946" border="0">

Figure 2. Impaired maturation of Fbn2-null osteoblasts. (A) Illustrative images of von Kossa staining of neonatal cOb (left) and adult MSCs (right) along with magnified van Geison–counterstained images (middle) of cOb differentiated for 21 d after OS administration. Histograms summarize the number of mineralized nodules in WT and Fbn2-null (Fbn2-/-) cOb (n = 15) and MSC (n = 5) cultures. Bars, 200 μm. (B) AP activity of WT and Fbn2-/- cOb measured 3 d before (day -3) and 4 d after (day 4) OS administration (day 0) and normalized to total protein levels (n = 5). (C) qPCR estimates of indicated transcripts in total RNA isolated from day 4 cOb cultures (n = 4; top) or P4 calvariae of WT and Fbn2-/- mice (n = 4; bottom). (D and E) MTT and BrdU incorporation assays (D) and C-myc and Ccnd1 mRNA levels (E) at the indicated days of cOb differentiation (n = 6). (F and G) Cell survival evaluated by Trypan blue exclusion (F; n = 4) and cell apoptosis (G) measured as the fraction of cleaved caspase-3 over full-length protein (with histograms representing densitometric analyses) in WT and mutant cObs. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 3. Osterix and collagen down-regulation in Fbn2-null osteoblasts. (A and C) Illustrative images of pOBCol2.3GFP (A) or Osx-GFP::Cre transgene expression (C) in Fbn2-null and WT cOb at day 17 or 4 of differentiation, respectively, with histograms summarizing the number of GFP-positive cells (n = 3); nuclei are DAPI stained. (B) Illustrative images of tibiae of Fbn2-/- and WT mice harboring the pOBCol2.3GFP transgene that include (from top to bottom) hematoxylin/eosin staining, GFP expression, and magnified views of toluidine- and GFP-positive cells; histograms summarize cell counts in the last images (n = 3). (D) von Kossa staining after 21 d of differentiation of cOb treated with or without 100 ng/ml rhBMP2 with histograms (right) summarizing numbers of mineralized nodules and levels of Osx and Col1a2 transcripts in mutant and WT samples (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 4. Fibrillin-2 controls TGF-β bioavailability. (A) Surface plasmon resonance sensograms of binding of immobilized L1K (left) and L4K (right) to rF86 at concentrations between 0 (baseline tracing) and 200 nM (top tracing). Binding was recorded as resonance units (RU), and nonspecific binding to control surface was subtracted at a molar ratio of 1:1. The table summarizes the affinity data (expressed as Kd) for interactions between the LTBPs and fibrillin-2 peptide. (B) Immunodetection of nuclear pSmad2 accumulation in day 4 cOb cultured in low serum with or without SB431542; nuclei are DAPI stained. Histograms summarize the percentage of pSmad2-positive nuclei in WT (black) and mutant (gray) cells (n = 3). (C) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn2-null cOb cultured in low serum (n = 3). (D) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn2-null cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (E) qPCR estimates of Tgf-β transcripts in WT and Fbn2-null cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 5. Elevated TGF-β signaling limits Fbn2-null osteoblast maturation. (A) Illustrative images of immunoreactive material corresponding to the indicated proteins deposited in the ECM of overconfluent WT, Fbn1-null, and Fbn2-null cOb cultures after 4 d of differentiation; nuclei are DAPI stained. Bars, 50 μm. (B) Maturation of WT and Fbn2-null cOb cultures treated with 1 μM SB431542, 300 ng/ml neutralizing pan–TGF-β antibody, or 50 μM Alk5 siRNA with histograms summarizing the number of mineralized nodules in each treatment (n = 3). (C) qPCR estimates of the indicated mRNA levels in the Alk5 silencing experiments. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 6. Abnormally high TGF-β activity in differentiating Fbn1-null osteoblasts. (A) Illustrative images of von Kossa–stained WT and Fbn1-null (Fbn1-/-) cOb after 21 d of differentiation with histograms summarizing the number of mineralized nodules in each sample (n = 5). (B) Cell proliferation of WT and mutant cOb at day -3 of cell culture as assessed by BrdU incorporation and qPCR quantification of C-myc and Ccnd1 transcripts (n = 3). (C and D) qPCR estimates of indicated transcripts in total RNA isolated from day 4 differentiating WT and mutant cOb cultures (C; n = 4) and from P4 WT and Fbn1-/- calvarial bones (D; n = 3). (E) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn1-null cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (F) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn1-null cOb cultured in low serum with or without 1 μg/ml of noggin (n = 3). (G) qPCR estimates of TGF-β transcripts in WT and Fbn2-null cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 7. Fibrillin-1 regulates BMP signaling in cultured osteoblasts. (A) Immunodetection of nuclear pSmad1/5/8 accumulation in day 4 differentiating WT and Fbn1-null (Fbn1-/-) and Fbn2-null (Fbn2-/-) cOb cultured in low serum; nuclei are DAPI stained, and histograms summarize the percentage of pSmad1/5/8-positive nuclei in WT (black), Fbn1-null (white), or Fbn2-null (gray) cells (n = 3). Bars, 25 μm. (B) Transcriptional activity of pBRE-lux reporter plasmid in day 4 differentiating WT, Fbn1-null, and Fbn2-null cOb (n = 5). (C) C2Cl2BRA bioassay measuring BMP signaling in conditioned media from WT, Fbn1-null, and Fbn2-null cOb and from the first two cell cultures treated with either 1 μg/ml noggin or 1 μM SB431542 (n = 3). (D and E) qPCR estimates of Bmp transcripts in day 4 Fbn1-/- and Fbn2-/- cOb cultures, respectively (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < id="p-3">

Figure 8. Microfibrils are not a structural substrate for matrix mineralization. (A) Illustrative von Kossa–stained WT cOb cultures in which Fbn1 or Fbn2 expression was silenced by RNAi with histograms on the immediate and far right showing the number of mineralized nodules and levels of indicated transcripts (n = 3), respectively. (B) Illustrative von Kossa–stained Fbn2-/- cOb cultures in which Fbn1 was silenced with histograms on the immediate and far right, showing the number of mineralized nodules and levels of indicated transcripts, respectively (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < href="http://4.bp.blogspot.com/_GjNXhZPfwZI/TNHCyOT0FhI/AAAAAAAAABM/Gxl-x2k602w/s1600/fig9.bmp">

DISCUSSION

In short, fibrillin-1 which is associated with MFS is shown to impair bone growth, as the absence of the related gene results in faster maturation than typical cells. On the other hand, the lack of fibrillin-2 is associated with CCA results in impaired "bone formation and osteoblast maturation." In the end, it can be said that the combination of fibrillins as well as their associated and binding proteins are responsible for both "anabolic and catabolic phases of bone homeostasis."

The TGF-β and BMP proteins are important in the specificity of the cellular signaling and resulting cascades. The combination of these two proteins is important only in the combined number of the two. The relative amounts in that sum has been shown to be unimportant. All of these effects, however, are said to be bone-specific after observing the effects of MFS and CCA. The details of this specificity is still unknown and needs to be further explored for a more comprehensive understanding of these protein effects and diseases.

METHODS

Experiments were done on both mice and cultured cells, ranging from no manipulation to absences of either the fibrillin-1 or -2 genes. The cultured cells were primary cultures of osteoblasts and mesenchymal stem cells (MSCs). RNA was collected with RNeasy Mini kits, and results were analyzed using immunostaining, qPCR, and a variety of bioassays (i.e. Surface Plasmon Resonance - SPR).

Critique

The conclusions drawn from the data of cultured cells were for endogenous activity; however, the experiments used exogenous stimulators/inhibitors. These in vitro conclusions are probably reasonably reliable however, as it is probably the activity levels rather than the activators themselves that matter. It would still have been nice if there was some way to implement an endogenous set of stimulators/inhibitors, so that the conclusions could be definite. On the other hand, the experiments on the modified mice did prove to be amongst the first to unambiguously show the importance of the fibrillin bindings and associations in the ECM for osteogenic differentiation. The combination of the findings from the cultured cells and modified mice also serve to reinforce the conclusions drawn from the in vitro experiments.

A couple of issues were not dealt with in these set of experiments. Firstly, the other cascades that could have resulted from high TGF-β activity were not accounted for. Any other resulting cascade could have influenced the observed effects or even produced some nuances. There is no reason to state that the observations and results are the effect of this single pathway. Another issue left untouched was the interactions between signaling events. Events occur in both the space and time dimensions and the signals are bound to have some sort of effect on each other. Spatiotemporal signaling events and consequences were not accounted for, and this something that should be looked into.

Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells

2010-11-01T15:35:00.000-07:00

http://www.nature.com/nature/journal/v293/n5830/pdf/293305a0.pdf

Nature 293, 305 - 307 (24 September 1981); doi:10.1038/293305a0

Gordon N. Gill & Cheri S. Lazar
Department of Medicine, Division of Endocrinology, University of California, San Diego, School of Medicine, La Jolla, California 92093, USA

Abstract

Epidermal growth factor (EGF), which binds to specific high-affinity cell-surface receptors, stimulates replication of a number of cell types1,2. In vitro EGF stimulates a membrane-associated protein kinase which catalyses phosphorylation of the EGF receptor at tyrosine residues3,4. The transforming proteins of several RNA tumour viruses are protein kinases which also specifically catalyse phosphorylation at tyrosine residues5−10. An elevated level of phosphotyrosine is found in cells transformed by Rous sarcoma, Fujinami sarcoma, PRCII, Y73, Snyder−Theilin and Gardner−Armstrong feline sarcoma and Abelson murine leukaemia viruses5,10−12. At least four of these viruses, which encode distinct protein kinases, catalyse phosphorylation of tyrosine residues in the same cellular substrate proteins13. In vitro EGF-stimulated protein kinase catalyses the phosphorylation of anti-p60src heavy chains, suggesting that this enzyme recognizes similar substrate determinants to p60src (refs 14,15). Here we demonstrate that EGF treatment of A431 human epidermoid carcinoma cells increases phosphotyrosine content, indicating that EGF stimulates tyro sine-specific protein kinase activity in vivo as well as in vitro. In contrast to Rous sarcoma virus (RSV) transformation, EGF inhibits replication of A431 cells. This inhibition by EGF is influenced by both cell density and tissue culture substratum.

Summary

A431 human epidermoid carcinoma cells have an unusually high density of EGF receptors. It has been known that EGF increases the phosphotyrosine content of exponentially growing A431 cells. EGF stimulates a membrane-associated protein kinase through phosphorylation. EGF increased the phosphotyrosine content of A431 cells sevenfold (0.14 and 0.02% in EGF-treated and untreated A431 cells respectively), indicating that EGF activates tyrosine-specific protein kinase activity in vivo.

Figure.1
Exponentially growing A431 cells were cultured. In control, no EGF was given while in EGF group, EGF was added. After culturing, cells were broken down and the proteins were collected. Electrophoresis was done and following Western blot was done and then overexposed to phosphoamino acids. The results shows phosphorylated tyrosine(p-tyr) only exists in EGF-treated cells.

Figure 2.
Effect of EGF on growth of A431 cells plated at different cell densities. Cells were cultured with different densities: 550(□), 2500(○) and 11000(●) cells / cm^2. The possibility that increased EGF degradation at high cell densities on tissue culture dishes contributed to the rightward shift in the dose-response curve shown in Figure 2.

Figure 3.
Effect of cell density on binding of EGF to A431 cells. A431 cells were subcultured into 3-cm tissue culture dishes and used 24-48 hr later. EGF-binding to A431 cells grown on tissue culture dishes: ●, I-EGF binding of constant specific activity. KD is the equilibrium dissociation constant. This figure shows that there is an inverse relationship between cell density and the affinity of EGF receptors for EGF.

Critique

Epithermal growth factor(EGF) is one of the molecules that cancer researchers are interested in. Many cancer cells have extraordinarily high density of EGF-receptors, and EGF is known to affect cell proliferation and apoptosis by phosphorylating EGF receptors.

This article is related to our groups project, whis is to see the effect of EGF on A431 cells in their morphology and proliferation. This research done by Gill and Lazar is one of the beginning researches of EGF on cancer cells. At the time of this research, the molecular activity of EGF on EGF receptor was figured out: in vitro, EGF was found out that it catalysed the phosphorylation of the EGF receptor at tyrosine residues. The main purpose of this research was to see the effect of EGF in vivo.

This article was published on Nature in 1981, so it does not show the current hot issues in molecular biology/bioengineering. However, it shows several interesting points that are worth taking a look. First, this research began as to see the effect of EGF in vivo, but as the experiment went on, the authors could discover a new experimental result that the effect of EGF was influenced by both cell density and tissue culture environment. This shows that even we start an experiment with a clear hypothesis and purpose, we may make unexpected but new discoveries.

Secondly, the methods used in this research are outdated and different from current methods. For example, if you look at Figure 1 which shows the protein electrophoresis result, you will easily see that the figure seems messy. The purpose of the electrophoresis was to visualize the amount of phosphorylated tyrosine in EGF-treated cells and control, so the dark background is actually a noise. Considering the fact the phosphorylated tyrosine had been studied well in vitro before this research was performed, I think there was a reason that they could not perform immunoblotting skills, which would have clearly shown the difference in amount of p-tyrosine. In my opinion, Western blot was not popular at the time of this research, because the Western blot was only invented in 1975, a few years before this research. Nevertheless, the researchers proved that there was significant difference in the amount of p-tyrosine by manually separating the proteins.

Lastly, this article does not have small titles as "introduction", "materials & methods", and "results" which can be found in most of today's articles. By reading an article like this, I realized that those small titles expedite the understanding of readers.

To sum up, this article is interesting because it is one of the first researches to see the effect of EGF in vivo. Even though it is from 1981, the skills and logic through the experiment are understandable, and it will let you have an opportunity to think about current research techniques in different way.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity

2010-11-01T14:57:00.004-07:00

Tsai, J., Lee, J.T., Wang, W. et al. (2008). Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad. Sci. U.S.A. 105, 3041–3046.

Background

Mutations in the BRAF gene occur in a wide variety of human tumors. Specifically, the BRAF V600E missense mutation is the most frequent oncogenic protein kinase mutation known. Tumor phenotypes correlated with oncogenic BRAF mutations include increased severity of cancer and decreased response to chemotherapy. One therapeutic approach to treating BRAF mutant cancer involves direct inhibition of the oncogenic BRAF kinase activity. Thus, in BRAF V600E mutants, targeted inhibition of the BRAF V600E gene product may be an important avenue to treating such tumors, especially in traditional therapy-resistant tumor types. The development of highly specific and effective inhibitors of the BRAF V600E gene product would provide insight into the therapeutic relevance of this target in malignant tumors. In this paper, the authors did exactly such by discovering the BRAF V600E specific inhibitor PLX4720 and examining its efficacy in both cell and animal based models.

Scaffold- and Structure-Based Discovery of the Inhibitor

The paper started off with a discussion of the rational drug design process that led to the discovery of PLX4720. Scaffold and structure based discovery was utilized. In other words, protein kinase scaffolds were first identified for a selected library of 20,000 compounds. The compounds were applied at 200 µM to multiple solved, but structurally divergent protein kinases. The compound-kinase structures were then screened by co-crystallography to identify which compounds would best bind to the known kinase scaffolds. A tremendous amount of work went into such screening process as over 100 structures showing bound compound were solved in this manner. Once productive binding activity was discovered for a given compound, chemical analysis was then performed to modify the compound for further inhibitory binding to the protein kinase. Through such a characterization process, PLX4720 was discovered to selectively bind to the BRAF V600E gene product (See Figure 1 for the PLX4720 binding structure and other structures leading up to its discovery). In fact, the authors claim, based on IC50 analysis (that is, the concentration at which 50% of the kinase is inhibited) PLX4720 binds with greater than 10-fold selectivity for BRAF V600E than for the wild-type BRAF.

Fig. 1. Structures of individual compounds leading to the discovery of PLX4720 are shown. (A) The chemical structure of 3-aminophenyl-7-azaindole (compound 1) is shown beneath its costructure with Pim-1 kinase. (B) The chemical structure of 3-(3-methoxybenzyl)-7-azaindole (compound 2) is shown beneath its costructure with the kinase domain of FGFR1. (C) The chemical structure of PLX4720 is shown beneath its costructure with B-Raf kinase.

Modes of Inhibitor Interaction and Selectivity

After characterizing the discovery process, the authors move on to further describe the binding action between PLX4720 and BRAF V600E. Crystallographic analysis revealed that there may be multiple conformations to the structure’s bound state. Some of these conformations reflect the “active” state of PLX4720, that is, the state in which the compound inhibits BRAF V600E, while other conformations reflect an “inactive” state. Such conformations and their respective binding interactions are displayed in Figure 2.

Fig. 2. Depiction of the three-dimensional structure of PLX4720 bound to B-Raf. (A) The structure of B-Raf^V600E bound to PLX4720 (yellow) is overlayed with an ATP model based on structures of ATP analogs in complex with other tyrosine kinases (orange). This view indicates that the PLX4720 scaffold overlaps with the adenine-binding site, but the tail of PLX4720 binds to a different pocket from the ATP ribose-triphosphate tail. The positions of the hinge, activation loop (A-loop), and phosphate-binding loop (P-loop) are also shown. (B) A surface representation shows PLX4720 binding to the B-Raf-selective pocket in the active conformation. (C) A surface representation shows PLX4720 binding to the kinase general pocket in the inactive conformation. (D) A close-up view shows the overlay PLX4720 bound to both active (green) and inactive (purple) conformations of the V600 protein, and PLX3203 (yellow) bound to V600E protein in the active kinase conformation. (E) A stereoview shows the specific interactions of PLX4720 to the active kinase conformation. In this conformation, the phenylalanine of the DFG loop is pointing in toward the compound-binding site. (F) A stereoview shows the specific interactions of PLX4720 to the inactive kinase conformation. In this conformation, the phenylalanine of the DFG loop is pointing away from the compound-binding site, and binding of PLX4720 is disfavored, leading to partial occupancy of this site even at the 1 mM compound concentration used in cocrystallography.

Cellular Selectivity in Multiple Tumor Lines

PLX4720 was then tested for its selectivity for BRAF V600E in multiple mutant and wild type cell lines. Each cell line was incubated for 1 hour in various concentrations of PLX4720 treatment. Two outputs were then measured, the GI50 (50% of growth inhibition) of cellular proliferation and GI50 of P-ERK expression. A previously characterized MEK inhibitor, PD0325901, was used as a control to benchmark P-ERK expression levels. It was also shown that PLX4720 is even more selective for BRAF V600E in cell lines than in kinase alone reactions. In some cases, the selectivity was up to over 100 fold. In addition, the clear down-regulation of P-ERK indicated that PLX4720 critically affected BRAF-MEK-ERK pathway. Notably, the BRAF-MEK-ERK pathway is generally regulated by feedback mechanisms in wide-type tumor cell lines, that is, even with BRAF inhibition, P-ERK is not critically down-regulated. Interestingly, this is not the case in BRAF V600E mutants, as demonstrated by the critical down regulation of P-ERK. The authors speculate that the negative feedback loop is lost in BRAF V600E cells, resulting in a pronounced dependence on the BRAF-MEK-ERK pathway.

Melanoma cell biology

To further drive the point home, the authors then demonstrated the effect of PLX4720 inhibition on a larger panel of melanoma cell lines. P-ERK expression was examined by Western blotting, and representative blots were shown in Figure 3A. Of the cell lines tested, 1204Lu (a BRAF V600E line) and C8161 (a BRAF wild-type line) were selected for further study because these cell lines come from highly malignant tumors that were resistant to chemotherapy. After administering varying concentrations of PLX4720 to these cell lines, it was demonstrated that 1204Lu displayed lower cell viability, while C8161 was unaffected (Figure 3B). This indicated that PLX4720 may have potential clinical applications where traditional chemotherapy has failed. In Figures 3C and 3D, an Annexin V PI stain coupled with cell flow cytometry was utilized to characterize 1205Lu and C8161 when treated with different concentrations of PLX or with the same concentration of PLX at different time points. The Annexin stains for alive vs. apoptotic cells, while the propidium iodide (PI) stains for necrotic vs. non-necrotic apoptotic cells. The results indicate that under equal PLX4720 treatment conditions, more cells in the V600E line underwent apoptosis than cells in the wide-type line. Again, this is consistent with the authors’ claims that PLX4720 selectively inhibits BRAF V600E. In Figure 3E, a live dead assay using Calcein-AM and EtBr was applied under varying treatment concentrations of PLX4720. Once again, it is shown that the BRAF V600E mutant line was more critically affected by PLX4720 and had more dead cells than the wild-type line. In Figure 3F and 3G, synthetic skin was created using 1204Lu (BRAF V600e) and C8161(BRAF wild-type) lines. The synthetic skin were respectively treated with PLX and immunostained with DAPI (for DNA), S100 (indication of cell proliferation), PCNA (indication of cell proliferation), and TUNEL (for DNA fragmentation). In all stains indicating cell growth and viability, it is shown that there is weaker signal in the PLX4720 treated BRAF V600E mutant synthetic skin, while DNA fragmentation is shown to be increased under PLX4720 treatment. Meanwhile, all 5 stains appear to be relatively unchanged after PLX treatment in the BRAF wild-type synthetic skin. This again served to drive the nail in the coffin in saying that PLX4720 selectively inhibits BRAF V600E lines.

Fig. 3. Selectivity and antimelanoma activity of PLX4720 in vitro. (A) Panel of melanoma cell lines (V600E+, left; B-Raf wild-type, right) were treated with various dosages of PLX4720, and protein extracts were subject to immunoblotting. Activity within the MAPK pathway is represented by levels of phosphorylated ERK; β-actin serves as a loading control. (B) B-Raf V600E+ (Left) and B-Raf wild-type (Right) cells were treated PLX4720 at the indicated dosages for 72 h. Cell number was assayed by MTT analysis. (C) 1205Lu and C8161 cells were treated with 1 μM PLX4720 for the times indicated and stained with Annexin V/FITC and propidium iodide (PI) for analysis of apoptosis. (D) Graphs represent raw data from the Annexin/PI assay. (E) Spheroids from 1205Lu and C8161 cells were treated with indicated dosages of PLX4720 and stained with calcein AM and ethidium bromide to assess overall viability. Green (calcein-AM) indicates live cells; red (EtBr) depicts apoptotic cells. (F) Synthetic skin was created by using 1205Lu (V600E+) cells and subjected to vehicle control (Upper) or 1 μM PLX4720 (Lower) for 72 h. H&E staining is depicted (Left), and immunofluorescent stains for DAPI, PCNA, and S100 are also shown. (G) Same as F, except with C8161 (B-Raf wild-type) cells.

Animal Efficacy

In addition to in vitro study, PLX4720 was further tested in mouse models for its efficacy to cause tumor regressions in vivo. A tumor xenograft of COLO205 cells (which are BRAF V600E mutant) was planted into nude mice. The xenograft mice then treated with either vehicle control, 5 mg/kg PLX4720, or 20 mg/kg PLX4720 by oral gavage daily on days 1-14 of the 25 day study. The results (Figure 4A) indicate that with increasing PLX4720 dosage, there was less tumor growth in the mice. In their style of driving the point home repeatedly, the authors then again performed xenografts using 1205Lu (BRAF V600E) and C8161 (BRAF wild-type) cell lines. Both were planted at two million cells into SCID mice, and the mice were treated with either vehicle control or 100 mg/kg PLX4720 by oral gavage twice daily. The results (Figure 4B and 4C) indicate that in the BRAF V600E tumors, PLX4720 caused significant tumor regression as there appeared to be little to no relative growth of the tumor, whereas in the BRAF wild-type tumor, PLX4720 appeared to have no effect. Furthermore, 1205Lu xenograft tumors were extracted from the mice, fixed in formaldehyde, paraffin embedded, and subject to immunostaining. Under immunostaining for P-ERK, the PLX4720 treated tumor extract indicated much lower P-ERK activity than the vehicle control. This indicates that PLX4720 not only selectively inhibits targets the BRAF V600E gene product in vitro (leading to tumor regression), but does so in vivo as well.

Fig. 4. Effect of PLX4720 on xenograft tumor growth. (A) Tumor volume measurements of COLO205 xenograft tumors treated with 5 or 20 mg/kg PLX4720 by oral gavage or treated with vehicle. Dosing occurred from days 1 to 14. (B and C) Two million cells [1205Lu (B); C8161 (C)] were s.c. injected into SCID mice. After reaching sufficient size, mice were treated by oral gavage with vehicle control (Left) or 100 mg/kg PLX4720 (Right) twice daily for the indicated times. (D) 1205Lu xenograft tumors were extracted, fixed in formalin, and paraffin embedded. Vehicle- (Left) and PLX4720- (Right) treated samples were immunostained for phospho-ERK.

Conclusion

In brief, the authors make the argument that PLX4720 (a compound they had discovered) selectively inhibits the BRAF V600E gene product. Upon inhibition, cell viability is decreased and tumor regression appears to occur in both cell and mouse models. This may have large clinical impact for cancer treatment as BRAF V600E is a common mutation known to occur in many cancers. This study may also shed light on the mechanisms of cancer inhibition as the BRAF-MEK-ERK pathway is shown to be critically down-regulated in BRAF V600E lines under PLX4720 treatment.

Critique

The authors of this paper went to great lengths to drive their point home through a variety of methods pointing to the same conclusion. This is not uncalled for, as the authors make a rather convincing argument using the substantial amounts of evidence obtained from several different experimental techniques. Readers are left convinced that PLX4720 appears to be a potent BRAF V600E inhibitor that will have a large impact on clinical cancer treatment. Indeed, this paper has been widely cited, and PLX4720 has been referred 4 times in the New York Times to date.

One thing to be desired from this study is justification of the concentrations and time-points at which PLX4720 was administered. The authors appeared to choose arbitrary values best suited to demonstrate their point. Not much thought had been given to the clinical relevance of such values. Since the end goal of PLX4720 is to be developed as an avenue of clinical cancer treatment, choosing clinically relevant concentrations is critical. For example, in the mouse model studies, the concentration of PLX4720 varied from as low as 5 mg/kg to as high as 100 mg/kg. There can be a significant difference in toxicity between such values. In future studies (perhaps closer to clinical phase II stage), it would be appropriate to address approximate toxicity levels for the inhibitor.

The use of the BRAF-MEK-ERK pathway to indicate inhibitor selectivity was never fully justified. It is only empirically observed that BRAF V600E mutants tend to be more dependent on the BRAF-MEK-ERK pathway. The authors provide some speculation such as failure of the negative feedback mechanism, but no evidence is ever provided. Moreover, the use of COLO205 over other V600E lines in mouse xenografts was also not justified. However, to be fair, justification of the BRAF-MEK-ERK pathway dependence is truly difficult and probably beyond the scope of this paper. Also, in the case of mouse xenografts, the authors supplement their study by using both BRAF V600E mutant and BRAF wide-type xenografts.

Cartilage engineered in predetermined shapes employing cell transplantation on synthetic biodegradable polymers

2010-11-01T14:32:00.000-07:00

Woo Seob Kim, Joseph P. Vacanti, Linda Cima, David Mooney, Joseph Upton, Wolfgang C. Puelacher, Charles A. Vacan. Plastic and Reconstructive Surgery, Vol. 94, No. 2, pp. 233-237. August 1994.

Introduction During plastic and reconstructive surgery, inorganic materials are often implanted to provide support to produce the desired aesthetic or reconstructive effect. However, these inorganic materials are often recognized as foreign bodies by the body's immune system, leading to infection and ultimate rejection. If cartilage is to be safely and successfully implanted in patients, it must be immune-capatible. In this study, a team of researchers and medical doctors designed biodegradable polymers in a variety of shapes (e.g. triangle, rectangle, cross, cylinder), seeded them with chondrocytes, and observed the specimens during a 12-week time course. The chondrocytes were observed to take on the shape of their scaffolds with little evidence of resorption by the body or overgrowth beyond the bounds of the scaffold. These results suggest that autogenous cartilage, developed from a patient's own cells and grown on a pre-designed scaffold holds promise for use in the fields of plastic and reconstructive surgery.

Materials and Methods Researchers began with an entanglement of polyglycolic acid fabric (PGA), whose fibers are randomly arranged. This was then submersed in a 2% (w/v) solution of poly-L-lactic acid (PLLA), which chemically bonds the fibers of PGA by forming cross-linkages. After removal from solution, the polymer constructs were cut into the desired geometric shapes: a triangle,a rectangle, and a cross.

Cartilage was harvested from a bovine source and washed in povidone-iodine 10% solution. Chondrocytes were subsequently harvested and concentrated to 5x107 cells per mL. 100uL of this solution was seeded into the scaffolds and supplemented with 10% fetal calf serum and an assortment of amino acids and antibiotics. Cell nutrients were labeled with BrdU to allow researchers to identify implanted cells. The cell-polymer constructs were incubated in 5% carbon dioxide at 37 degrees Celsius, with the culture medium replaced every 3 days. After a week, the cell-polymer constructs were subcutaneously implanted into the dorsum of mice. For the cylindrical cartilage scaffolds, sheets of the polymer construct were cut into rectangles and seeded with chondrocytes that were incubated for a week. These polymer-cell constructs were then wrapped around 2.5 cm long silicone rods of 3mm diameter before being subcutaneously implanted into the dorsum of mice. After 3, 6, 9, and 12 weeks, mice were sacrificed and specimens evaluated with histological stains. Results and Discussion Gross examination of the non-cylindrical scaffolds (12 triangles, 12 rectangles, 12 crosses) showed that all scaffolds were replaced with chondrocytes that retained approximately the same shape as the scaffold. Two of the 12 cylindrical constructs became infected and were extruded through the skin -- the other 10 remained subcutaneous and showed replacement be chondrocytes, with retention of scaffold shape.

Histological staining showed that cells found on the periphery of the implants were more disordered and less mature than those located centrally, which exhibited deeper basophilic staining that suggests increased mucopolysaccharide concentration characteristic of mature cartilage. Immunohistochemical staining found BrdU labeling in the periphery of the cartilage, suggesting that cells located in this region were produced after in-vitro culturing. By week 3, the polymer fibers of the polymer-cell implants had begun degrading, with degradation continuing throughout the 12-week time course of the study.

Critique While this study represents a promising advance in creation and manipulation of autologous cartilage constructs, there are still several problems that must be addressed before this technique becomes feasible for use in human patients. To begin with, the time course of this study is relative short (12 weeks), whereas most cartilaginous implants are expected to last the duration of a patient's life. It is unclear if the shape of the cartilage may change past the 12 week mark; if the cartilage was to begin to lose its shape or lose its structural integrity, these implants would no longer be appropriate for use in plastic and reconstructive surgery. Researchers must also address the mechanical properties of cartilage formed on these scaffolds -- do they have the structural integrity of naturally-occuring cartilage? Can the mechanical properties be varied as a function of experimental parameters (e.g. density of injected cells, growth medium conditions)? This is an important aspect to address because cartilage used in different parts of the body may require different mechanical properties. A third problem the researchers may consider addressing is the immunoreactivity of their implants. It is interesting to note that in this study, chondrocytes harvested from calves were injected into scaffolds that were then implanted into mice. No mention of immune reactions is noted besides the two cylindrical scaffolds that were extruded. It is unclear if this immune reaction is due to the calf chondrocytes or perhaps the scaffold. Though 12 weeks is conceivably sufficiently long for an immune response to be elicited and noticed, long-term effects are unlikely to be noticed during the time course of this study. In conclusion, this paper presents a novel, perhaps revolutionary way for cartilaginous implants to be used in plastic and reconstructive surgery. However, before human implantation becomes even remotely feasible, researchers and medical doctors must take into account a host of factors that may influence the feasibility of such a design.

Tissue engineered autologous bladders for patients needing cystoplasty

2010-11-01T13:14:00.000-07:00

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1B-4JN2NY2-2&_user=4420&_coverDate=04/21/2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1575238480&_rerunOrigin=scholar.google&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=6d860a6cd88e8d22f5943ba9bc04b6c8&searchtype=a

http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(06)68438-9/fulltext

Anthony Atala, Stuart B. Bauer, Shay Soker, James J. Yoo, and Alan B. Retik

The Lancet, Volume 367, Issue 9518, Pages 1241 - 1246, 15 April 2006

Introduction and Background

Cystoplasty is a medical procedure used to surgically repair aspects of the bladder. It is commonly needed when medication is not enough to maintain organ function. Patients with myelomeningocele often require cystoplasty, as they experience frequent urination as often as every half hour. Traditionally, repair has been achieved by using two sources. Synthetic sources such as polyvinyl sponge, Teflon, collagen matrices, and, and biological sources such as the skin, omentum, placenta, and small intestines have been tested for use in cystoplasty. However, tissue grafts have often failed due to mechanical, structural, functional, and biocompatibility issues. Atala and his group have been working on a new system of reconstructing bladder tissue using the patient’s own cells. Their technique involves seeding scaffold with urothelial (bladder) and smooth muscle cells, incubating the construct in vitro, and implanting the organ into the patient.

Materials and Methods

The study and surgical technique was performed on 7 patients (range 4-19 years old) with severe bladder complications due to myelomeningocele. For each patient, urothelial and muscle cell samples were taken and cultured in vitro using standard techniques. Two types of scaffolds were made, one comprised of a decellularized bladder submucosa and the other consisting of a collagen-PGA composite. The bladder scaffolds were custom-made to match the sizes of the patient’s original organs, all with thickness at around 2 mm. The scaffolds were seeded with about 700 x 10⁶ cells of each type. The exterior was seeded first with muscle cells, incubated for 1 hour, and submerged in media that was changed every 12 hours. Next, the interior was seeded with urothelial cells, and the entire construct was incubated in media for 3-4 days when the surgery began.

During the procedure, the patients’ bladders were cut open, and the scaffold was oriented and attached in its place with fibrin glue and polyglycolic sutures. Some patients also had an omental wrap surrounding the construct. Urinary catheters were inserted during the surgery to allow for urine drainage during post-operative recovery. After a three week period, the bladder was conditioned by a cyclic process of clamping and draining through the catheters. Post-operative tests were done that included histological staining of the bladder, serum analyses, ultrasounds, leak point pressures, cystograms, and other urodymanic tests.

Results and Discussion

All the patients tolerated the procedure with no significant complications. Overall, bladder compliance and maximum leak-point pressures (indicating storage capacity) increased, showing a significant improvement with the engineered bladders. Histological tests confirmed proper growth of urothelial and muscle cells. The group found that the optimal technique was the use of a collagen-PGA construct that was inserted with an omental covering.

Critique

This study shows great promise in tissue engineering. The group has been testing tissue regeneration from autologous sources for quite a while, and the success of the group’s technique on human patients yields major implications for the future of tissue engineering. The relatively simple structure of the bladder even allowed them to tackle the challenge at an organ-level, but dealing with other organs will surely be more challenging.

The paper introduced many variables that were monitored, like the use of decellularized bladder submucosa versus a collagen-PGA scaffold, the effect of an omental wrap on bladder function, and the time intervals for cyclic conditioning of the engineered bladder. Performed with only 7 patients, there should be more replicates to fully determine which combination of techniques is optimal for bladder reconstruction. Also, although it showed an improvement in compliance and leak-point pressures, it fails to reference the ideal values that are typical for fully functional bladders. Moreover, the paper did not strongly address long-term factors related to the procedure. For example, it failed to mention the degradation behavior of the collagen-PGA scaffold, nor does it analyze the appropriate vascularization of the system. Thus, I believe more tests can be done to fully analyze the technique of autologous bladder reconstruction.

Tissue-Engineered Lungs for in Vivo Implantation

2010-11-01T11:11:00.000-07:00

Thomas H. Petersen, Elizabeth A. Calle, Liping Zhao, Eun Jung Lee, Liqiong Gui, MichaSam B. Raredon, Kseniya Gavrilov, Tai Yi, Zhen W. Zhuang, Christopher Breuer, Erica Herzog, Laura E. Niklason1,

Introduction:

Human lungs usually do not repair beyond the cellular level, and currently the only method of replacing damaged lung tissue is through lung tissue transplantation, an expensive procedure that achieves less than 20% survival rate after ten years. Techniques to decellularize organs and use them as an acellular matrix scaffold to generate lung tissue have recently been tried with promising results. However, regenerating viable lung tissue is a tall order as the tissue should: contain lung specific cells, have branching structure of airways, separate blood and air, and allow for respiration. In an attempt to construct functional lung tissue using a rat model, lung tissue was decellularized and the resulting scaffold was repopulated with neonatal lung epithelial cells. The populated lung scaffold was cultured in a bioreactor simulating fetal lung environment. Lastly, the lung was tested for functionality.

Materials and Methods:

Preparation of a decellularized lung scaffold

Lung tissue from adult Fischer 344 rats was harvested and treated with detergent solution for roughly 2-3 hours. The resulting lung matrices, as verified by micro-CT scans indicate an intact microcellular matrix. The protocol was effective in preserving the micro structural properties of the native lung, yet removal of any antigenic components that may interfere with culturing of tissues.

Properties of the lung bioreactor

A bioreactor was used to culture the cells on the acellular scaffold. Cells were injected into the vasculature and airway compartments, to which they adhered. Subsequent tests indicated healthy proliferation and low incidences of apoptosis. Culture medium was fed though the pulmonary artery at physiological pressures and a negative pressure breathing loop with air was implemented to simulate respiration. The breathing loop was essential for ensuring cells did not block the airways and cleared the airways of any obstructions. In addition, exposure to air increased the numbers of epithelial cells, which was one of the essential criteria for a functional lung tissue graft. Compliance tests yielded high similarities between native tissue and engineered tissue, and an immunohistochemical staining indicated the seeded endothelial cells were extensively distributed with tight junctions between them. It was noted that extended culture periods resulted in a cellular distribution more similar to that of native lung tissues. In order to check if such scaffolding techniques are also compatible with human tissue, human lung tissue underwent a similar protocol of decellularization to obtain a matrix that was repopulated with lung cells. The cells adhered to the vasculature suggesting that such techniques may be compatible with human lung tissue, but an extensive study was not performed.

Implantation of engineered lungs into rats

The cultured engineered lungs were implanted and replaced the left lungs of four animals. Within minutes blood flow was established in the engineered lung vasculature. X-rays showed that inflation in the engineered lung was less than that of the right native lung. Additionally minimal bleeding was observed.

Results:

Data collected primarily consisted of testing of the engineered lung properties before testing for ability for gas exchange. Compliance testing for the elasticity of the engineered lung yielded similar stress-strain values between the native lung and the engineered lung. However, it was noted that the engineered lung was noticeably less elastic than the native lung; however, the difference was considered minor and did not alter the performance of the engineered lung.

Implantation of the engineered lung – Blood gas analysis of the blood taken from the engineered lung indicated that oxygen and carbon dioxide gas exchange did occur. Partial pressure of oxygen increased from 27 +/- 7mmHg in the pulmonary artery to 283 +/- 48 mmHg in the left pulmonary vein indicating hemoglobin oxygen saturation. However, this partial pressure is quite different from the native lung’s (634 +/- 69 mmHg. Additionally, levels of carbon dioxide exchange fell from 41 +/- 13mmHg in the pulmonary artery to 11 +/- 5 mmHg in pulmonary vein indicating efficient carbon dioxide removal.

Discussion

Overall, performance of the engineered lung was quite promising, although the tissue could not perfectly emulate that of native tissue. The engineered tissue overall was not as pliable and did leak some blood. However, performance was sufficient for the oxygenation of blood. Despite this, the success of the engineered tissue demonstrates a strong feasibility in the use of micro architecture of native lung tissue for regeneration of functional lung tissue. Still, many issues must be overcome for long term engineered function to persist. Ideally, the air-blood barrier must be improved to prevent further bleeding and production of certain proteins and surfactant needs to be increased. Lastly, other technologies for obtaining compatible epithelium such as lung stem cells is still required to make this a feasible clinical procedure.

Critique

The focus of this paper was to test the efficacy of the use of their engineered lung tissue as a functional tissue transplant. Considering that such functional lung tissue needs to emulate so many properties of native lung tissue in order to even function, and then for these researchers to be able to create such a working model is quite impressive. Based on the performance of the tissue, the researchers laid out clear goals to tackle in the future for improving the efficiency of the tissue. Aside from the mentioned technical improvements for tissue function, potential future projects include culturing of human lung tissues and improving incubation techniques.

However, there are several minor points in the paper that could be improved. Based on the protocol, a total of four engineered lungs were implanted into four rats. Because the researchers were simply testing the efficacy of the transplant, this number is adequate, although small. Future, improved engineered lungs could be tested with more rat models. Testing with human lung tissue was briefly mentioned but whether the cells could actually be cultured was not pursued. Given that this was not the purpose of the experiment, more tests need to be done before assessing whether human lung tissue could truly be created via similar techniques. Additionally, the researchers mentioned that the engineered lungs were functional for short periods of times, up to two hours, but little additional information was given on why this time frame was chosen. Whether the rats died or were simply euthanized after a period of time or that the tissues quickly lost efficiency is unknown. Lastly, much more qualitative data may be provided in terms of lung tissue properties. A small plot for the stress-strain characteristics of the tissues is provided albeit lacking details. However, the mention of reduced surfactant production and protein expression such as aquaporin-5 could be paired with relevant data.

Reprogramming of human somatic cells to pluripotency with defined factors

2010-11-01T10:30:00.000-07:00

In-Hyun Park¹, Rui Zhao¹, Jason A. West¹, Akiko Yabuuchi¹, Hongguang Huo¹, Tan A. Ince², Paul H. Lerou³, M. William Lensch¹ & George Q. Daley¹

Abstract

Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.

Introduction

Pluripotency is the property of cells to different into all tissues of an organism. Once differentiated from the embryonic stem cells, adult human somatic cells cannot differentiate into other types of cells. Induced pluripotency involves transfecting genes that can dedifferentiate cells into the pluripotent state. These induced pluripotent stem cells (iPSCs) have many of the same properties as Human embryonic stem cells (HSCs). Park et al. attempted to use the four main reprogramming factors to isolate iPS cells from differentiated H1-OGN cells: OCT4, SOX2, KLF4, and MYC. Undifferentiated cells of the H1-OGN line were differentiated and tested for expression of these four factors over a prolonged time period. This showed that only expression of the Myc gene persisted after 5 days of differentiation as all other expression quickly died down.

In the human ES cell line H1-OGN⁸, the OCT4 promoter drives expression of GFP-IRES-neo. a, Time course of differentiation of H1-OGN cells into a population of adherent fibroblasts, and subsequent expansion of a colony into a clonal fibroblast cell line (dH1cf32). The differentiated fibroblast derivatives of H1-OGN cells are morphologically indistinguishable from dermal fibroblasts cultured from an adult volunteer donor (hFib2). b, Quantitative real-time PCR demonstrates that the expression of a cohort of key pluripotency factors (OCT4, SOX2, NANOG and KLF4) is lost by the third week of differentiation, whereas expression of a fifth factor (MYC) persists.

Methods

Several experiments were performed, which involved cell culture as well as gene expression analysis. Cell culture was complicated by the use of specific factors and human ES cell medium to allow for formation of iPS colonies. Retroviral production involved introduction of OCT4, SOX2, KLF4, and c-MYC via the pMIG vector. The viral infections lasted for 24 hours and were subsequently seeded onto MEFs for five days. Chromosome counts confirmed that cell lines were diploid. Microarray analysis was performed by isolating RNA, preparation of RNA probes for microarray hybridization, and then scanned and analyzed. Assay for Teratoma formation involved injection of resuspended iPS cells into mice at a cell concentration of approximately 1 x 10⁶ cells.

Reprogramming of human ES-cell-derived fetal fibroblasts

The differentiated cell line dH1cf was then infected with a lentiviral cocktail that had human OCT4, SOX2, Myc, and KLF4. A week later, cells were plated in HeSC culture. Two weeks after infection, small colonies were seen that were picked and expanded, which resulted in more colonies that were identical to parental H1-OGN cells. Morphology was decided to be a sufficient marker, without need for selection with G418, as seen in previous literature with murine iPS cells. Ten independent transfections of 10⁵ dH1cf cells resulted in about 100 cells exhibiting ES-cell morphology, which is a low efficiency of about 0.1%. More interestingly, it was observed that ES-cell like colonies were still formed when Myc and Klf4 were eliminated from the viral cocktails, but the resulting efficiency was much lower.

Figure 2: Multiple cultured human primary somatic cells yield iPS cells.

a, iPS cells produced from five independent human primary cell lines form colonies with a similarly compact, ES-cell-like morphology in co-culture with mouse embryonic feeder fibroblasts (MEFs). b–f, As shown via immunohistochemistry (IHC), human iPS cell colonies express markers common to pluripotent cells, including alkaline phosphatase (AP), Tra-1-81, NANOG, OCT4, Tra-1-60, SSEA3 and SSEA4. 4,6-Diamidino-2-phenylindole (DAPI) staining indicates the total cell content per field. Fibroblasts surrounding human iPS colonies serve as internal negative controls for IHC staining. dH1f-iPS3-3 (b, from H1-OGN differentiated fibroblasts), MRC5-iPS2 (c, from MRC5 human fetal lung fibroblasts), BJ1-iPS1 (d, from neonatal foreskin fibroblasts), MSC-iPS1 (e, from mesenchymal stem cells), hFib2-iPS2 (f, dermal fibroblast from healthy adult male).

High resolution image and legend (364K)

Table 1: ES-cell-like colony formation with various donor cells and reprogramming factors

Reprogramming of fetal, neonatal and adult fibroblasts

A diverse panel of human primary cells was tested with viral transfection containing the four factors (OCT4, SOX2, Myc, and KLF4) in an effort to dedifferentiate these cell lines into an iPS state. Fetal lung fibroblast cells (MRC5) and fetal skin cells (Detroit 551) formed human ES-cell like colonies after introduction of the four aforementioned factors. However, this method did not work for all of the cell lines, including neonatal foreskin fibroblasts, adult mesenchymal stem cells, and adult dermal fibroblasts.

The researchers were able to add additional factors, which eventually resulted in human ES-cell like colonies. These additional factors were suspected to be necessary for the cells to be grown in continuous cell culture and reprogramming to pluripotency. These genes were hTERT (catalytic subunit of human telomerase) and SV40 large T (anti-apoptotic). When these genes were transfected along with the four original pluripotency factors, there were some human ES-colonies, despite there still being significant cellular loss.

Results - Characterization of reprogrammed somatic cell lines

The isolated colonies of reprogrammed iPS cells were analyzed in several ways. Immunohistochemistry showed expression of alkaline phosphatase, Tra-1-81, Tra-1-60, SSEA3, SSEA4, OCT4, and NANOG – markers characteristic of human ES cells. Quantitative PCR studied the gene expression of the derivatives, showing that the following genes were markedly more expressed: OCT4, SOX2, NANOG, KLF4, hTERT, REX1, and GDF3. These expression levels were comparable to the parental H1-OGN human ES cells.

Although hTERT and SV40 large T genes were used in addition to the four genes to form a six-factor viral cocktail that produced iPS cells for postnatal cell lines, these genes were not expressed by the ES cell-like colonies isolated from the reprogramming. These genes may still be useful indirectly in cell culture to increase the efficiency of reprogramming, but they are not intrinsically essential for the viral transfection.

Global messenger RNA expression analysis was performed on H1-OGN cells, parental fibroblast cells, and reprogrammed iPS derivatives. Cluster plots were formed using the Perason correlation. Analysis of the scatter plots shows that there is a tighter correlation between reprogrammed cells and human ES cells than between differentiated fibroblasts and human ES cells, or differentiated fibroblasts and human iPS derivatives. Therefore, the iPS cells isolated from somatic sources are highly similar to the embryonic human stem cells at the global transcriptional level.

Finally injection of pluripotent cells into mice and subsequent teratoma formation has become a standard way to test pluripotency. The iPS cell derivatives caused cystic tumors in mice that exhibited all three primary germ layers, demonstrating the pluripotent ability of the isolated iPS cells. This result, in addition to the previous analysis involving gene expression and comparison to parental Human ES cells, suggests that the derived human ES-cell like colonies in this experiment are indeed induced pluripotent stem cells.

Figure 3: Gene expression in human iPS cells is similar to human ES cells.

a–e, Quantitative real-time PCR assay for expression of OCT4, SOX2, NANOG, MYC, KLF4, hTERT, REX1 and GDF3 in human iPS and parental cells. Individual PCR reactions were normalized against internal controls (β-actin) and plotted relative to the expression level in the parent fibroblast cell line. a, dH1f, dH1f-iPS3-3, dH1cf16-iPS-1 and dH1cf32-iPS-2 cells. b, MRC5-iPS2, MRC5-iPS12 and MRC5–iPS17. c, BJ1-iPS1. d, MSC-iPS1. e, hFib2-iPS2 and hFib2-iPS4. f, Transgene-specific PCR primers permit determination of the relative expression levels between total, endogenous (Endo) and retrovirally expressed (Transgene) genes (OCT4, SOX2, MYC and KLF4) via semi-quantitative PCR. β-Actin is shown as a positive amplification and loading control.

High resolution image and legend (257K)

Figure 4: **iPS cells are demethylated at the OCT4 and NANOG promoters relative to their fibroblast parent lines.**

Bisulphite sequencing analysis of the OCT4 and NANOG promoters in H1-OGN human ES cells, dH1f differentiated fibroblasts, dH1f-iPS-1, dH1cf32-iPS2, as well as the MRC5 neonatal foreskin fibroblast line and its derivatives MRC5-iPS2 and MRC5-iPS19. Each horizontal row of circles represents an individual sequencing reaction for a given amplicon. White circles represent unmethylated CpG dinucleotides; black circles represent methylated CpG dinucleotides. The cell line is indicated to the left of each cluster. The values above each column indicate the CpG position analysed relative to the downstream transcriptional start site (TSS). The percentage of all CpGs methylated (% Me) for each promoter per cell line is noted to the right of each panel.

High resolution image and legend (265K)

a, A Pearson correlation was calculated and hierarchical clustering was performed with the average linkage method in H1-OGN, dH1f, dH1f-iPS3-3, dH1cf16, dH1cf-iPS cells (dH1cf16-iPS5 and dH1cf32-iPS2), MRC5, MRC5-iPS2, BJ1 and BJ1-iPS1 cells. The distance metric calculated by GeneSpring GX7.3.1 for comparisons between different cell lines is indicated above the tree lines. The fibroblast lines dH1f, dH1cf16, MRC5 and BJ1 cluster together, whereas iPS cells cluster together with the H1-OGN human ES cell line. b, Global gene expression patterns were compared between differentiated fibroblasts (dH1f, dH1cf16), reprogrammed somatic cells (dH1f-iPS3-3, MRC5-iPS2) and human ES cells (H1-OGN). Red lines indicate the linear equivalent and twofold changes in gene expression levels between the paired samples.

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Figure 6: Xenografts of human iPS cells generate well-differentiated teratoma-like masses containing all three embryonic germ layers.

Immunodeficient mouse recipients were injected with human iPS cells (dH1f-iPS3-3) intramuscularly. Resulting teratomas demonstrate the following features in ectoderm, mesoderm and endoderm. Ectoderm: pigmented retinal epithelium (a), neural rosettes (b), glycogenated squamous epithelium (c); mesoderm: muscle (d), cartilage (e), bone (f); endoderm: respiratory epithelium (g). Of note, panel c contains all three germ layers: (1) glycogenated squamous epithelium, (2) immature cartilage, (3a) glandular tissue with surrounding stromal elements, and (3b) another small gland. All images were obtained from the same tumour. Tissue sections were stained with haematoxylin and eosin. Scale bar, 100 μm.

High resolution image and legend (353K)

Conclusions:

This paper shows a method to induce pluripotency in several different cell types, including adult human somatic cells. The results establish feasibility in using methods similarly used for iPS reprogramming of murine cells and performing these transformations for human cells, a big step towards clinical application of these findings. OCT4 and SOX2 have been determined to be the main components of dedifferentiation, however Klf4, Myc, and other factors such as SV40 large T and hTERT can greatly increase the efficiency of this process. Before clinical success with human iPS cells can occur, there must be a developed method to avoid genetic transmutation by the lentivirus, which is nonspecific at this time. Future work would combine studies of targeted viral transfection as well as iPS factors for a predictable dedifferentiation therapy.

Critique:

This prominent study, published in Nature, is a milestone in stem cell research, because it was one of the first iPS stem cell papers that isolated the genes necessary for reprogramming to iPS state using multiple adult cell types. The four transcription factors changed to induce pluripotency were: Oct4,Sox2,Klf4, and Myc. The methods used in the study include retroviral transfection, cell culture, bisulphite genomic sequencing, and microarray analysis. The interesting phenomenon observed was that only Oct4 and Sox2 were absolutely necessary to induce pluripotency, while the other genes increased the efficiency of stem cell colony formation. This is significant, because it isolates the specific transcription factors that result in pluripotency, which is an important discovery for this type of research. Also, the study is one of the first to use human somatic cells instead of mouse somatic cells, demonstrating that iPS cell formation is possible. One problem is that the isolation of the four factors could have been more rigorous, because it was based on previous research for mouse somatic cells, so there could have been factors that were not considered. The only controls used were internal negative controls, and the researchers did not use positive controls in their microarray analysis. Finally, the quantification of iPS cell formation was not specific and is subjective. A better method of quantification should be developed instead of checking for "cell-like morphology." Overall, this paper does a good job of approaching iPS research with the significant goal of manipulating various types of adult human somatic cells.

Supplementation of Fat Grafts with Adipose-Derived Rengenerative Cells Improves Long-Term Graft Retention

2010-11-01T07:33:00.001-07:00

Min Zhu, MD, MS, Zhengyu Zhou, BS, Yan Chen, MD, MS, Ronda Schreiber, PhD, John T. Ransom, PhD, John K. Fraser, PhD, Marc H. Hedrick, MD, Kai Pinkernell, MD, and Hai-Chien Kuo, PhD.

Introduction

The use of adult stem and progenitor cells is emerging as a novel therapeutic option for many diseases: adipose-derived regenerative cells (ADRCs) include several types of stem and regenerative cells, inculding adipose-derived stem cells (ADSCs). ADSCs have the capacity to differentiate into multiple cell lineages and secrete factors that are angiogenic, antiapoptotic, and pro-adipogenic. The objective of this study was to determine whether ADRC supplementation could improve the long-term graft retention and survival in a mouse fat transplantation model.

Materials and Methods

Murine ADRCs were isolated from the fat pads of ROSA26 mice after 6-12 weeks, through washing, mincing, digestion with collagenase I, and centrifugation. Total RNA was isolated from ADRCs and analyzed using RT-PCR for genes of interest, including mouse VEGFA, HGF, IGF-I, and GAPDH. Inguinal fat pads were isolated, mixed with either saline or a solution of ADRCs, and injected subcutaneously into the skulls of female mice. 6 and 9 months after transplantation, the animals were killed and the grafts were weighed and prepared for hematoxylin and eosin staining, beta-gal staining, and immunohistochemistry. Morphometric analyses were performed to evaluate intact and nucleated adipocytes, cysts and vacuoles, fibrosis, and capillary density. LacZ-postive ADRCs were identified using X-gal. Immunohistochemistry and immunocytochemistry were performed using antibodies directed against CD31, CD34, and CD144.

Results

ADRCs formed extensive tubule-like structures after reaching confluence when plated. These structures showed the presence of for endothelial cell markers, suggesting that ADRCs include cells producing pro-angigogenic factors and extracellular matrix. PCR analysis showed high levels of expression in angiogenesis-related genes.

FIGURE 1. ADRCs form an extensive tubule-like network in vitro. ADRCs form tubule-like structures in DMEM+ 10% FBS in the absence of coated extracellular matrix and additional growth factors. These capillary-like structures were positively stained for endothelial cell markers: CD31 (A), CD34 (B), and CD144 (C). All the images were taken at 100× magnification.

FIGURE 2. ADRCs express angiogenesis-related genes. A comparison between ADRCs and BM-MNCs shows ADRCs highly express many angiogenesis-related genes as compared with BM-MNCs. A relative gene expression ratio above 1 indicates a given gene is expressed more in ADRCs than in BM-MNCs; actual values are noted above each bar. Gene array comparisons were performed twice, showing a similar pattern of relative gene expression, one of which is shown here (A). A comparison of ADRCs to fibroblasts, BM-MNCs, and PBMNCs using real time RT-PCR confirms that ADRCs abundantly express VEGFA, HGF, and IGF-1. For the ease of data presentation, gene expression in each cell type is compared with fibroblasts here. The number above or below each bar indicates the fold of gene expression change in each cell type relative to fibroblasts with a positive value indicating gene up-regulation and a negative value indicating gene down-regulation (B).

Mice transplanted with fat and ADRCs showed greater fat retentention on the skull, with the weight of grafts with ADRCs being about twice that of grafts with only fat at both 6 and 9 months. Histologic examination showed that grafts that included ADRCs had significantly more intact and nucleated adipocytes and fewer cysts and vacuoles than those with only fat. These grafts also had a higher capillary density, as shown by the immunostaining of CD31.

FIGURE 5. ADRCs improve the quality of fat grafts and increase capillary densities within the grafts. Representative H&E sections of fat grafts harvested at 6 months showed that the grafts in the Fat+ADRCs group (B) contained more adipocytes and fewer cysts/vacuoles than those in the Fat-only group (A). Immunostaining of CD31 showed that the grafts in the Fat+ADRCs group (D) had a higher capillary density than those in the Fat-only group (C). CD31⁺ endothelia cells exhibit a brown color, whereas the blue color is due to nuclear staining with methylene blue. All the images were taken at 200× magnification.

FIGURE 6. Incorporation of ADRCs into vessel walls in fat grafts. A representative histologic section of fat grafts harvested at 6 months. Immunofluorescence staining of CD31: CD31⁺ endothelial cells were stained in red (A). [beta]-gal staining:LacZ⁺ cells were stained in blue (B). LacZ⁺ cells were found in vessel walls (arrow). The 2 pictures were taken from the same tissue section. All the images were taken at 200× magnification.

Discussion

Supplementation of fat grafts with ADRCs can improve the long-term retention and quality of grafts, mediated at least in part by the ability of ADRCs to increase neovascularization within the grafts. The presence of ADSCs in vessel walls and the presence of pro-angiogenic and anti-apoptotic factors suggested that ADRCs promote angiogenesis through paracrine signaling. A cooperative interaction between multiple types of stem and regenerative cells in ADRCs may enhance graft survival and quality through several mechanisms.

The clinical utility of ADRC-enhanced fat grafting has been tested in cosmetic breast augmentation and breast reconstruction with positive results. The breat reconstruction trials showed a statistically significant improvement in mean tissue thickness measurement and no significant loss of benefit within twelve months. Previous results also showed that ADRCs did not promote tumor formation when coimplanted with different types of breast cancer cells in SCID mice. Overall, these studies suggest that ADRC therapy is feasible and likely to be safe.

Limitations of this study include the the harshness of the environment in which the fat grafts were placed (the skull of the mice), which led to a low baseline level of graft retention. Another limitation is that the study cannot distinguish between fat graft retention and endogenous adipose regeneration. In conclusion, this model shows that the supplementation of fat grafts with ADRCs can improve the long-term retention and quality of the transplant, an effect mediated by multiple mechanisms that include angiogenesis, with implications for clinical breast augmentation and reconstruction procedures.

Critique

It seemed that authors pushed analysis in the direction of angiogenesis and vasculogenesis as the main mechanism through which the presence of ADRCs improves graft retention. The results did not seem to be very strong regarding the presence of adipose-derived stem cells in vessel walls in fat grafts, and in fact the authors describe it as a “rare event.” This point was addressed in the discussion of the paper, as the authors admitted that there were multiple mechanisms taking place and that the low presence of ADSCs suggested angiogenesis as opposed to vasculogenesis. Effectively, I was given the impression by the way this paper was written that when the authors did not find data that strongly backed up their assumptions, they were not very willing to reconsider these assumptions.

The presentation of data regarding histologic staining was also slightly difficult to read. An explanation in tabular format of the scores used as measurements, as opposed to written explanations, would have been more clear.

Overall, this study succeeds at building upon previous research in tissue engineering to further the possibilities for clinical application of stem cells and progenitor cells. It is clear that the authors are focused on addressing issues related to tissue engineering, such as immunogenicity (solved in the approach of using autologous cells) and the possibility of tumor formation (a different study conducted by the same authors and referred to in the discussion).

Conditional telomerase induction causes proliferation of hair follicle stem cells

2010-11-01T01:18:00.000-07:00

Kavita Y. Sarin, Peggie Cheung, Daniel Gilison, Eunice Lee, Ruth I. Tennen, Estee Wang, Maja K. Artandi, Anthony E. Oro & Steven E. Artandi

Introduction

Telomerase, or more specifically, its protein component TERT, is traditionally known to maintain the length of telomeres. Through this mechanism, it plays a role in the increased proliferative capability of stem cells, progenitor cells, as well as cancer cells. Sarin et. al., however, found that TERT can promote the proliferation of resting stem cells through a non-canonical pathway. They used the conditional transgenic induction of TERT to promote robust hair growth in mouse epithelium. They found that TERT causes a rapid transition between telogen (the resting phase of the hair follicle cycle) and anagen (the active phase). More specifically, TERT overexpression stimulates the proliferation of quiescent stem cells in the hair follicle bulge region. This function does not require the RNA component of telomerase, which codes for telomere extension, and thus operates through a mechanism independent of its traditional function of maintaining telomere length.

Methods and Results

Sarin et. al. obtained protein extracts from wild-type mouse skin, and analyzed the first and second postnatal hair cycles using the telomere repeat amplification protocol (TRAP assay). They detected strong telomerase activity during the anagen phases, and none during the telogen phases (Fig. 1b).

a, A schematic diagram of hair follicle cycling. b, TRAP on non-transgenic (Non-Tg) skin samples at day 4, 10, 16, 19, 21, 28, 34 and 52 (anagen, A; catagen, C; telogen, T). c, d, Northern blot analysis and TRAP on skin extracts from i-TERT and wild type (WT) mice at day 50. e, Doxycycline-treated mice at day 70, Non-Tg (+ doxy) and i-TERT (+ doxy) mice. f, H&E skin sections at 20 magnification. -doxy indicates no doxycycline treatment; asterisk indicates telogen hair follicle; arrow indicates anagen hair follicle. g, RNA in situ hybridization for TERT mRNA and immunofluorescence for K14 in i-TERT (+ doxy) skin (asterisk indicates autofluorescence). h, RNA in situ hybridization for TERT mRNA (blue) in an i-TERT(+ doxy) skin section (right panel) and WT anagen skin section (left panel) (asterisk indicates dermal papilla).

To determine whether TERT expression can induce a transition from telogen to anagen phase, they injected induced TERT (i-TERT) mice with doxycycline after their hair follicles had entered the telogen phase (Fig. 2), and took tissue samples at regular intervals for 12 days. TERT mRNA and telomerase activity were found to increase from day 3 to day 9 (Fig. 2a).

a, Northern blot (left) and TRAP assay (right) show increased TERT expression and telomerase activity after nine days of doxycycline treatment. b, H&E stain showing that follicles in i-TERT mice entered anagen (black arrows) by day 9, whereas Non-Tg controls remained in telogen (asterisks). c, Hair growth was observed only in i-TERT mice with doxycycline treatment (+ doxy), but not in i-TERT mice (- doxy) or Non-Tg littermates.

Since the activation of stem cells in the bulge section of hair follicles is critical to the initiation of anagen cycles, they hypothesized that TERT’s effects on the hair follicle cycle are mediated through stem cells. To test this hypothesis, they marked hair follicle bulge stem cells with repeated injections of BrdU followed by a long chase period. During the second telogen, the mice were biopsied, switched to doxycycline (a TERT inducer) drinking water and biopsied again between days 80 and 100. Label retaining cells (LRCs) were visualized by double immunostaining with antibodies against BrdU and CD34, a cell surface marker for hair follicle stem cells. LRCs were present in similar numbers in both i-TERT and non-transgenic mice at day 55, before the switch to doxycycline containing water.
After five weeks of doxycycline treatment, the BrdU label in CD34 stem cells was retained at similar levels in non-transgenic mice. In contrast, the BrdU label was significantly weakened in the CD34 cell population in the bulge in i-TERT mice (Fig. 3a, b). Despite the loss of the BrdU label, the CD34 cells remained in similar numbers in the bulge, suggesting that stem cells divide but likely self-renew to maintain the CD34 population when exposed to TERT. The conclusion is drawn that TERT causes hair follicle bulge cells to proliferate, diluting the BrdU label from the quiescent stem cell population.

a, Immunofluorescence for BrdU (red) and CD34 (green) shows maintenance of LRCs in non-transgenic (Non-Tg) group, but dramatic loss of label in i-TERT mice after doxycycline (doxy) treatment (pre-doxy, day 55; post-doxy, day 90). Asterisk indicates autofluorescence of hair. b, Quantification of LRC data from a, showing the fraction of CD34⁺ cells that are also BrdU⁺. Data for i-TERT mice (black bars, n = 4 mice) and non-transgenic mice (grey bars, n = 3 mice), pre-doxy (- ) and post-doxy (+ ) treatment are shown. c, LRC analysis from wholemounts of epidermis from the tail of mice labelled with BrdU at day 10, switched to doxy at day 40 and analysed at day 65 (BrdU, red; K14, green); B indicates bulge and SG indicates sebaceous gland. d, Immunofluorescence using Ki-67 (red) to mark proliferating cells and K14 (green) to identify basal layer of skin. e, Quantification of proliferation index in d as Ki-67⁺ cells per 100 m length of basal layer (each comparison n = 2 mice). i-TERT mice (black bar) and non-transgenic mice (grey bars). f, GFP epifluorescence co-stained with CD34 (inset, confocal microscopy) in skin section from an actin–GFP mouse. g, RNA in situ analysis for TERT mRNA in i-TERT(+ doxy) mouse skin (inset, TERT mRNA expression (cytoplasmic) overlaps in bulge with LRCs, marked by BrdU (nuclear)). h, H&E sections from K5tTA⁺;tetop-TERT⁺(- doxy) (bottom) and Non-Tg (top) mice, at 20 magnification. Error bars indicate standard deviation. P values derived from Student's t-test.

To establish the theory that TERT in this case operates through a non-canonical pathway (that is, without TERC, the RNA component of telomerase), they intercrossed TERC +/- mice with i-TERT alleles. They found that conditional activation of TERT induced anagen in five out of five i-TERT+, TERC- mice (Fig. 4a).

a, H&E sections showing TERT-induced anagen in mice with TERC^+/+, TERC^+/- and TERC^-/- backgrounds, at 20 magnification. b, Skin samples from i-TERT TERC^-/- mice (+ doxy) lacked telomerase activity by TRAP (left panel) and lacked TERC expression by RT–PCR (right panel). Minus sign indicates negative control lacking reverse transcriptase.

Critique

Sarin et. al. demonstrate an effective examination of their hypothesis. Using TRAP, Northern blotting, immunofluorescence, and tissue analysis, they provide multiple methods to create a thorough support of their conclusion. They also considered possible side effects, and created additional experiments to account for them. For instance, they considered whether abnormalities in the hair follicle cycle could be causing the altered phenotype. Overall, they convincingly showed that TERT causes the proliferation of hair follicle stem cells, and that a rapid transition from telogen to anogen phase results.

There are just a few minor weaknesses in their paper. First, there ideally should be more than five mice used to demonstrate the non-canonical pathway of TERT. Also, in a few cases, they make assumptions. The words “probably” and “likely” show up in isolated situations, such as when they say that the stem cells “probably self-renew to maintain the CD34 population”. Obviously, not all assumptions can be supported by today’s experiments, but this is just something to watch out for.

Electroporation of polymeric nanoparticles: an alternative technique for transdermal delivery of insulin

2010-11-01T00:24:00.000-07:00

Rachna Rastogi, Sneh Anand and Veena Koul

Introduction:
There have been various techniques developed for delivery of drugs such as insulin into the body. Transdermal delivery of peptides and proteins has drawn interest since the technique allows control of drug input rate relieves patients’ fear of needles. But penetrating the skin barrier, stratum corneum (SC) creates a problem for this technique. Nanosized vesicles made with block copolymers of poly(ε-caprolactone) (PCL) and polyethylene glycol (PEG) are loaded with insulin and delivered to rat abdominal skins (in vivo and in vitro) through application of high voltage and current (electroporation). Studies are then done on the alterations of the skin structure and changes in the blood glucose level to see the effectiveness of the technique.

Methods:
Synthesizing the vesicles:
The copolymer was synthesized in a tri-block orientation: PCL-PEG-PCL. The polymers were then emulsified with 1 mg insulin. Formulation was stirred until nanoparticles are formed. Nile red (NR), a type of dye, was loaded into the vesicles to allow better detection.

Electroporation protocol:
The electroporation process requires a stainless steel pin electrode array clamped to a square wave pulse generator. Effects of voltage, pulse length and number of pulses are determined by insulin flux rate. Depth of penetration by the vesicles is detected by fluorescence microscopy (seeing the NR).

In vitro experiment:
Franz diffusion cells, systems composed of receptor and donor cells, are placed on a magnetic stirrer. The insulin-loaded nanovesicles are the donors while the rehydrated rat skin acts as the receptor. Pulses are then introduced to the skin to induce insulin transfer. The insulin concentration was determined by a solid-phase two-site immunoassay (similar to Western Blot). The depth of the nanovesicles is detected using confocal laser scanning microscopy (CLSM).

In vivo experiment:
Male adult Wistar rats are induced with diabetes mellitus and tested with subcutaneous injection of insulin as control. Gel contained with nanovesicles loaded with insulin is then applied on the rats followed by pulsing with pin electrodes. Blood glucose level was monitored and serum insulin level was determined using enzyme-linked-immunosorbent assay (ELISA).

Results and Discussion:
Polymeric vesicles, similar to phospholipid vesicles, can passively permeate through the transdermal membrane, but at a slow rate. Application of electrical current (electroporation) is believed to speed up the transfer rate. Polymeric vesicles were made with insulin entrapment efficiency of around 25-40%.

Insulin flux was measured under variable voltage, number of pulses, and pulse length. Passive diffusion of insulin was negligible. Insulin flux changes linearly with increase voltage. The rate of permeation remains elevated several hours after electroporation, suggesting that the prolonged change in permeability may be caused by altered skin structure. A linear increase is also observed with increase in pulse number and pulse length. The max flux of insulin was observed with 10 pulses lasting 15 ms at 100V.

Figure 3. (A) Effect of pulse voltage, (B) number of pulses, and (C)
pulse length on electroporative insulin flux across rat skin. A good
linear trend was observed with pulse voltage (R2 = 0.9514) and pulse
length (R2 = 0.9937); n = 4 for all experiments.

Histological assessment:
The SC was seen to be detached from the underlying layers in all electroporated samples. Application of electroporation also showed widening of the openings of hair follicles and an increase in ruffling of the epidermis. Recovery of the skin structure at 24 to 48 hours were studied and shown in the figure.

Figure 4. (A) Histological examination of untreated rat abdominal skin. Hair follicles showed separation and increase in pore size as marked.(B) The hair bulbs were also dilated. (C) The stratum corneum (SC) surface showed formation of ‘craters’ or pits with stripping SC layers. Extent of recovery of treated skin at (D) 24 and (E) 48 hours postelectroporation.

Passive treatment vs. electroporation:
Application of electroporation shows about 6.5 times increase in cumulative amount of insulin permeated compared to passive vesicles. The sustained permeation of insulin after the electroporation shows that the vesicles are intact after pulsing. Figure 5. Top: Comparison of passive and electroporative application
of loaded vesicles on the permeation of insulin across rat skin.
Bottom figure shows the quantities of insulin retained in the skin
after electroporation of solution and vesicles after 24 hours.

Assessment by CLSM:
The depth of penetration was dependent on the duration of contact with the electrode. A max depth is reach at 60 μm in 24 hours. Vesicles were present throughout the complete depth of the tissue. Study of Blood Glucose Level (BGL):
Electroporation technique of delivering insulin mimics the blood glucose profile of the IV injection. A slight delay is observed in electroporation due to the slow movement of vesicles across the skin layer. Therefore, compared to IV injection, the effect does not reach the blood circulation instantaneously. Also, electroporation of the nanovesicles has a longer delivery period, giving a possibility for patients to reduce the frequency of administration.
Figure 7. Reduction in blood glucose levels (%) in diabetic Wistar
rats on injectable and electroporative administration of insulin and
insulin-loaded nanoparticles [Control, saline i.v.; insulin (s.c.), subcutaneous
insulin; NP (i.v.), i.v. administration of insulin-loaded
nanoparticles; insulin (electro), electroporative delivery; NP (electro),
electroporation of insulin-loaded nanoparticles]. Inset shows
initial hypoglycemia posttreatment till t = 6 hours. All results
expressed are a mean of 6–8 animals.

Conclusion:
Electroporation of nanovesicles can be considered comparable to direct injection as shown by the in vitro and in vivo experiment. In vitro experiment demonstrated that the vesicles penetrate the deeper layers of epidermis while the in vivo experiment shows a prolonged hypoglycemic level up to 36 hours. Further studies need to be done on the effect of vesicle degradation and whether related toxicity may be involved.

Critique:
The paper has showed in depth study on nanoparticle deployment of insulin through electroporation with both an in vivo and in vitro experiment. The in vitro experiment was to demonstrate that the particles are able to penetrate disperse through the layers of the skin and into the bloodstream. The vivo experiment was to show that the blood glucose level can be regulated by the insulin delivered. The results from the tests on the various electroporation parameters seemed slightly suspicious since all three different parameter showed a linear increase, but the paper did mention that other papers have shown similar results. Also, the paper mention that the efficiency of trapping the insulin within the nanoparticle is around 25-40%, yet it does not mention a method to insure that the nanoparticles delivered through electroporation actually contains insulin. Most figures and graphs in the article are easy to interpret except the last one concerning blood glucose level. There were too many components in the graph, which made it hard to comprehend.
Further studies definitely need to done before this technique can be used since the paper itself stated that mouse skins are not good test subjects since it differs greatly from human skins. Also, as mentioned in the conclusion, effects of the byproducts from the degradation of the nanopaticles are still unknown.

Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells

2010-11-01T00:19:00.000-07:00

Kathyjo A. Jackson, Susan M. Majka, Hongyu Wang, Jennifer Pocius, Craig J. Hartley, Mark W. Majesky, Mark L. Entman, Lloyd H. Michael, Karen K. Hirschi, and Margaret A. Goodell

Introduction

The blockage of a blood vessel eventually results in myocardial ischemia, a condition that promotes rapid myocardial necrosis of the heart and scar tissue formation. Consequent re-perfusion of the vessel and heart repairs the ventricular wall to only a limited capacity. Cardiac myocytes cannot replicate, and the ventricular walls are replaced by scarring. Without treatment, ischemic heart muscle pumps blood and oxygen less efficiently, and may lead to heart failure and stroke.

This study seeks to explore the potential of adult stem cell therapy for repairing ischemic heart muscle. The researchers transplanted side population (SP) cells, enriched hematopoietic CD34^- stem cells, into mice rendered ischemic by coronary artery blockage. SP cells were shown previously to regenerate cardiac myofibers and blood vessels in injured cardiac tissue. Subsequent measurements of cardiomyocyte and endothelial cell differentiation were taken based on donor marker identification (lacZ gene expression).

Materials and Methods

Bone marrow specimens were extracted from the tibias and femurs of 6-Rosa transgenic mice, which express lacZ. To isolate SP cells, which account for 0.05% of whole bone marrow, the specimens underwent Hoechst dye staining and specific protein expression—most SP cells are positive for the markers c-Kit and PECAM-1. A triple-laser instrument was used to sort the desired SP cells through fluorescence emission analysis; the purity of SP cells in the sorted samples always exceeded 91%.

2,000 SP cells isolated from male 6-Rosa-Ly-5.2 mice were injected into irradiated female 6-Ly-5.1 mice. 10 weeks after the stem cell transplantation, the left anterior descending coronary artery of each mouse was blocked for 60 minutes, and followed by reperfusion. Two or four weeks after the injury, the hearts were removed and frozen for immunohistochemical analysis.

Frozen sections of tissue were fixed with paraformaldehyde and stained for lacZ expression. Specific antibodies were used to identify cardiac muscle (anti-α-actinin) and endothelial cells (anti-ICAM-1). Hematopoietic cells were visualized using anti-Ly-5-biotin. Slides of stained tissues were analyzed with fluorescence and differential interference contrast microscopy. SP cells and their progeny could be detected through lacZ expression, since they were derived from the 6-Rosa transgenic mice, which express lacZ.

Results

22 mice received bone marrow transplants; the coronary artery blockage procedure was performed on 19 of the mice—the remaining 3 mice acted as the control, where no procedure was done. After the hearts were sectioned, lacZ staining existed mostly in the capillaries. Co-staining with antibodies against Flt-1 and ICAM-1 illustrated that the SP cells had migrated to the injured heart through the circulation, localized to new vessels, and integrated into the surface linings (Figure 1). There were no lacZ-positive cells in the control mice.

Figure 1—Sections incorporated into vascular endothelial cells. (a-d): X-gal stained tissue for SP cell incorporation. (e-l): cardiac tissue as above, but stained for endothelial marker expression (Flt-1 and ICAM-1).

LacZ-positive cells were also incorporated into cardiac muscle; cardiac tissue from treated mice included staining throughout the heart. LacX-positive cells clustered at the edge of the myocardial scar, where most of the damage resides. Figure 2 illustrates the distribution of lacZ staining within cardiac tissue.

Figure 2—LacZ staining accumulates at the border of the myocardial scar. (a): X10 photograph of a mouse myocardial infarction after 4 weeks. The arrow points at the lacZ staining location. (b): X20 pictograph of the same lacZ area. Open arrowheads indicate macrophages. (c) X40 pictograph. (d) macrophage density after 1 hour of ischemia and 3 hours of perfusion.

Table 1 shows the array of lacZ-positive cells in various mouse subjects. 100 sections from each animal were analyzed, and the degree of lacZ staining was quantified. Each section contained about 13,000 cardiomyoctes; there was a mean engraftment of 0.02% for SD cells of all cardiomyocytes. 100 vessel profiles were analyzed per tissue section in terms of cell engraftment. The mean prevalence of lacZ-positive vessels was about 3%.

Table 1—Table illustrating the percent engraftment for cardiomyocytes and endothelial cells with regards to lacZ cell presence. Cardiac tissue analyses were conducted on 4 mice.

Discussion

Because myocardial infarction is a major cause of heart failure, new sources of cells are needed for transplantation and heart muscle regeneration. SP cells represent a progenitor population both accessible from a patient’s own tissue and adaptable for clinical use. This study demonstrates the ability of purified stem cells derived from mouse bone marrow to participate in cardiac muscle regeneration following ischemia. The SP cells marked with the lacZ gene migrated into cardiac myofibers, and aided in the regeneration process. In addition, the study showed that lacZ-positive cells could participate in vascularization within heart tissue. The utilization of SP cells into the regeneration of endothelial cells and cardiomyocytes suggests the role of circulating stem cells for tissue repair.

Critique

The study demonstrates the therapeutic potential of SP cells for cardiac tissue regeneration. Clinical trials on mice do indeed show incorporation of stem cells into the heart’s scar formation, as well as new endothelial tissue. The research does a good job in establishing a control group to see the effect of SP cells on a healthy mouse heart. Unfortunately, many limitations exist within this study. The researchers rely on lacZ expression as the major identifier of migrating SP cells. Other identifiers such as surface markers and morphology could have been obtained to establish a more reliable identification method. The study analyzed only 4 mice with regards to heart tissue, due to the large number of mice that died after the coronary artery procedure. A larger sample size may reveal better results. In addition, the amount of SP cell engraftment was relatively low for both cardiac muscle and endothelial tissue. Further studies involving SP cells and the tissue regeneration rate of live mice would be helpful.

Development of a Porcine Bladder Acellular Matrix with Well-Preserved Extracellular Bioactive Factors for Tissue Engineering

2010-11-01T00:05:00.000-07:00

Bin Yang, M.D., Ph.D.,Yifen Zhang, M.D.,Liuhua Zhou, M.D.,Zeyu Sun, M.D.,Junhua Zheng, M.D.,2 Yun Chen, M.D., Ph.D.,1 and Yutian Dai, M.D., Ph.D.1

Intro

Regenerative medicine builds upon the utilization of a specifically engineered scaffold for the growth of different cell types. These scaffolds’ functionality lies in providing an environment for cells similar to the endogenous environment of the tissue. This allows proper growth and protein expressions of these cells to ensure continued functionality over time once it is in the patient. There have been a number of various synthetically derived scaffolds however the complexity of the ECM is incredibly great making these approaches quite difficult. Another approach is the decellularization of xenogeneic tissues to use as a scaffold for tissue regeneration. However there is variability in methods to decellularize tissues and little research has been done on porcine urinary bladder decullarization. Yang et al explore a porcine bladder acellular matrix while considering the efficiency of decellularization and preservation of certain important biological factors within the matrix.

Methods

The decellurization of the porcine bladder tissue was done in 4 different ways for comparison to identify the best method. The first was Group A, which was cut into small pieces and the urothelium/suburothelium was removed surgically. They were then incubated overnight with ice-cold hypotonic Tris-EDTA buffer. Then they were washed with ice cold PBS and placed in a room temperature hypertonic Tris buffer containing Triton X-100 for 24 hours. Group B was similar to Group A except after the final incubation, this group was further incubated at room temperature in a hypertonic solution of Tris buffer containing SDS(sodium dodecyl sulfate). Group C was also similar in terms of reagents used for the incubation steps in Group A, except Group C was kept intact and distended for the duration of the incubation. Group D was similar to Group C except the Tris buffer contained less Triton X-100.

All of the groups were then treated with RNases and DNases and mechanically agitated in order to remove the cells. The tissues were subsequently tested for a number of biological factors including collagen, which was analyzed by the chloramine-T method. Total genomic DNA was also measure and compared with native porcine bladders using a fluorescence microplate reader. Then the active biological factors in the matrix were evaluated by using two different cell lines, HMSMC, and HUVEC. Both were cultured in DMEM with 10% FBS and maintained in a incubator with media exchange every day and were used at passage 2-4. The HBSMC was seeded at 4x10^3 cells/well and the HUVEC was seeded at 5x10^3 cells/well. Cell migration was also tested. VEGF and PDGF was also quantified through use of an ELISA kit in order to determine the extracellular bioactive molecules.

Results and conclusion

Histological specimens of the different groups were compared and after DAPI staining it became evident that in groups B and D there was total elimination of the cellular nuclei. Also since the amount of DNA detected was significantly lower for group D than A, it implies that the distension when preparing the acellular matrix caused improved efficiency in decellularization. In addition, it seemed that the group C scaffold better promoted the proliferation of the cells. Furthermore, total collagen was greater in group C than in group B. It seems the distension of the bladder thinned the walls and made removal of cell and other immune responsive components easier

. A combination of treatment with Triton X-100, distension, and buffer treatment seemed to be the best approach for effective decellularization. Also the total content of collagen was higher in the decellularized matrices compared to that of native porcine tissue. It seems that used of Triton X-100 left the proteins in the matrix intact allowing better cellular adhesion and proliferation.

Commentary

Overall the paper is interesting and applicable in the realm of tissue engineering. It provides insights into the difficulty of engineering matrices suitable for tissue regeneration since a number of factors can alter the state of the matrix. In addition, the matrix needs to be properly decellularized in order to remove immune responsive elements however; this must be done carefully to preserve bioactive factors that will promote cellular adhesion and proliferation when attempting to regenerate tissue. My main critique is questioning their ability to state that they preserved these necessary bioactive factors by looking at only a select few factors. They looked at collagen and sulfated GAG however these may not be the best representation of actual bioactive factors necessary for angiogenesis and tissue production. In addition, it is difficult to tell whether or not these bioactive factors supposedly remaining on the surface of this matrix will cause an immune response or not within a human model. Since it is a xenogeneic matrix, the proteins and molecules might be in a slightly different three-dimensional configuration, which could then be immune rejected by the body. However, it seems their work is decently compelling and provides better research into methods of decelluarization and it would be interesting it method similar could be developed to obtain similar results for other various tissue applications.

A nonviral minicircle vector for deriving human iPS cells

2010-10-31T23:55:00.000-07:00

Journal: Nature Methods

Publish Date: Feb 7, 2010

Authors: Fangjun Jia1, Kitch

ener D Wilson1,2, Ning Sun1,

Deepak M Gupta3, Mei Huang1, Zongjin Li1,

Nicholas J Panetta3, Zhi Ying Chen4, Robert C Robbins5,

Mark A Kay4 , Michael T Longaker3,6 & Joseph C Wu 1,6

1Departments of Medicine and Radiology, 2Department of Bioengineering, 3Department of Surgery, 4Departments of Pediatrics and Genetics, 5Department of C

ardiothoracic Surgery and 6Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

Summary:

In this work, the authors demonstrate a new technique to reprogram human adipose stem cells (hASCs) into Induced Pluoripote

nt Stem Cells (iPSCs) with the use of a plasmid like "minicircle DNA" vector. The importance of this work is a demonstration of using none viral vectors to reprogram cells. Reprogramming by viral vectors (pioneered by Shinya Yamanaka), may be carcinogenic since the host genome is altered by the insertion of viral vectors, thus, therapeutic applications are limited. The authors show that the minicircle DNA vector generates the protein transcription factors for reprogramming but does not integrate into the host genome itself, which implies that this method may be a safer technique for therapeutic applications.

The minicircle DNA vectors are similar to plasmids, in this case they are GFP tagged, however they do not have a bacteria replication origin and antibiotic resistance gene. Once electroporated into hASCs, four reprograming t

ranscription factors (Nanog, Oct4, Sox2, Lin 28) and a GFP signal can be observed. Since these minicircle vectors are do not replicate itself in the cell, they are diluted once cells divide.

qPCR, RT-PCR,Southern Blotting, was done to show the generation of reprogramming transcription factors. Bisulfite pyrosequencing was used to confirm a low methylation state. Imunostaining was performed to confirm stem cell markers, and morphology was also confirmed to be similar to iPSCs.

Critique:

Currently iPSCs generation efficiency with Yamanaka viral vectors are approximately 0.01%, however, with this technique, the efficiency is only at around 0.005%. I believe one of the major reasons for this low efficiency is the minicircle delivery method- electroporation. Although electroporation can deliver the vectors effectively, it lowers the viability of the cells greatly. This is due to the lack of control on the electrical field environment around each cell.

Since reprograming needs a continuous dose of the minicircle DNA (for the generation of the transcription vectors), they choose to use lipofectamin for subsequent delivery of the minicircle DNAs, which may have caused low

er delivery efficiency. Thus, i believe, if there was a novel minicircle delivery system which has higher delivery efficiency for the minicircle vectors, it would be a major factor that may be able to boost the overall reprogramming efficiency.

Figures in the paper.

Fig. 1-a=> shows GFP decreases as cell divides, and transcription factors are generated.

Fig. 1-b,c,d=> shows stem cell markers during immunostaining

Fig. 1-e=> RT PCR shows reprogramming genes being active.

Fig. 1-f=> methylation is reduced after transfection.

Fig. 1-g=> heat map comparison

Fig. 1-h=> southern staining

Figure 1 | Generation of iPSCs with minicircle vector. (a) FACS and qPCR analysis of hASCs after transfection with the minicircle vector on day 0. Percentage of GFP positive cells (left) and transcript fold change (right) are plotted. Error bars, s.d. (n = 3). (b) Brightfield (left) and fluorescence (right) images of a day 18 cluster of minicircle-derived iPSCs (mc-iPSCs). (c,d) mc-iPSCs stained for alkaline phosphatase (c) and immunostained for pluripotency markers (d). Scale bars, 100 ␣m (b,d) and 500 ␣m (c). (e) Reverse transcription–PCR (RT-PCR) analysis of three iPSC subclones (iPSC-1s–3s) derived from three separate donors; H9 hESC line; hASCs; and 293-MC negative control (293FT cells 24 h after transfection with the minicircle vector). Endogenous (endo) OCT4, SOX2, NANOG and LIN28 were analyzed. RN18S1 is 18S RNA. (f) Bisulfite pyrosequencing measuring methylation in the promoter regions of OCT4 and NANOG in the indicated cells. Distances upstream of transcription start sites (TSS) are indicated in base pairs. (g) Heatmap of microarray data (top) and scatter plots depicting gene expression fold changes between paired cell types (bottom; the iPSC data are the average of subclones 1 and 2). Highlighted are OCT4, SOX2 and NANOG expression (arrows). Green lines indicate fivefold changes in expression between samples. (h) Southern blot analysis of genomic DNA from the indicated cell lines using probes for OCT4 and SOX2. Lenti-iPSC, lentivirally reprogrammed iPSCs as positive control for genomic integration. MC-EcoRI, minicircle vector after digestion with EcoRI; MC-undigested, undigested minicircle vector.

Figure 2 | Pluripotency of mc-iPSCs. (a) Reverse transcription–PCR (RT-PCR) analysis of undifferentiated (U) and differentiated (D) mc-iPSCs for pluripotency markers (OCT4, NANOG and REX1) and various differentiation markers for the three germ layers (ectoderm, SOX1 and PAX6; mesoderm, T and KDR; endoderm, SOX17 and FOXA2. (b) Phase- contrast images showing cell types differentiated from mc-iPSCs. (c) Subcutaneous injection of mc-iPSCs caused teratomas in severe combined immunodeficient (SCID) mice. Teratomas consisted of all three embryonic germ layers, including neural tissue (neuro.; ectoderm), cartilage (mesoderm) and glandular structures (endoderm). Representative tissue sections from subclone mc-iPSC-1s are shown. Scale bars, 500 ␣m (b) and 100 ␣m (c).

BE115's Tissue Engineering Blog

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Reprogramming of human somatic cells to pluripotency with defined factors

Nature 451, 141-146 (10 January 2008) | doi:10.1038/nature06534; Received 16 November 2007; Accepted 10 December 2007; Published online 23 December 2007

Methods

Reprogramming of human ES-cell-derived fetal fibroblasts

Figure 2: Multiple cultured human primary somatic cells yield iPS cells.

Table 1: ES-cell-like colony formation with various donor cells and reprogramming factors

Figure 3: Gene expression in human iPS cells is similar to human ES cells.

Figure 4: iPS cells are demethylated at the OCT4 and NANOG promoters relative to their fibroblast parent lines.

Figure 6: Xenografts of human iPS cells generate well-differentiated teratoma-like masses containing all three embryonic germ layers.

Conclusions:

Critique:

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Development of a Porcine Bladder Acellular Matrix with Well-Preserved Extracellular Bioactive Factors for Tissue Engineering

A nonviral minicircle vector for deriving human iPS cells

Figure 4: **iPS cells are demethylated at the OCT4 and NANOG promoters relative to their fibroblast parent lines.**