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

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="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgPj1kD5z84xd0aK2J6vx-AQxnycSmFfx0oE1m2GRTo2m9dBwBUxFs-AvCrlTY-NNjG9Z5aDhLEze54jSiYMlnqNMofWhxw8W-0YN1QrlsvkFIBqWUbh5LrsEEm5AWbYa2zPKcn/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="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjDeAk42Bq4q3eP7jMCQcWz9gL_SOkNd5F9Eh800aXmrwE5oFq1TxpUGjF1w-qC8uEf9hC7aIaaTo1y2EIT_HN_rjSIoPKhUaX4_yY-4IX49neG3JiBi60SY7U0hgobqKvAN4Eq/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

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.

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

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

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

OR

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

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

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

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).

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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.

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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.

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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.

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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.