Gene Expression by Fibroblasts Seeded on Small Intestinal Submucosa and Subjected to Cyclic Stretching

Thomas W. Gilbert, Ann M. Stewart-Akers, Jennifer Sydeski, Tan D. Nguyen, Stephen F. Badylak, and Savio L-Y. Woo

Introduction
Previous research has demonstrated that porcine-derived small intestinal submucosa extracellular matrix (SIS-ECM) scaffolds can be successfully manipulated and applied for tendon and ligament repair in preclinical animal trials. In vivo experiments show that if the SIS-ECM scaffold is subjected to a site-specific mechanical environment, the healing response is improved, allowing the scaffold to promote the formation of site-specific tissue. As a result, the scaffold remodeling process reduces the incidence of scar tissue. The aim of this study is to characterize the response of fibroblasts seeded on SIS-ECM due to mechanical loading. This was accomplished in vitro by examining the changes in expression of matrix-related genes that would be predictive of the in vivo remodeling process and by correlating these changes with alterations in mechanical behavior of the SIS-ECM during stretching experiments.

Materials and Methods
For this study, a cyclic-stretching tissue culture (CSTC) system was specifically developed to independently measure in real-time the mechanical load of each scaffold. Additionally, the system applies specific displacement waveforms to each scaffold without disruption of the cell culture environment. This CSTC system consisted of eight independently operating stations, with each station containing a culture chamber, tissue clamps, a linear actuator and a load cell (Fig 1).

Figure 1. (A) Photograph of the CSTC system with the independent operating stations. (B) Representation of a single operating station, containing a linear actuator, sterile stretching chamber and a load cell to monitor load generated by the scaffold.

Porcine small intestine was harvested from market-weight pigs immediately after euthanasia. The SIS-ECM was isolated, de-cellularized in a 0.1% peracetic acid/4% ethanol solution, and rinsed in phosphate buffered saline and de-ionized water. SIS-ECM scaffolds were then rehydrated in modified DMEM and cut into fragments 4 cm in length and 0.8 cm in width. Each scaffold was seeded with 1.6 x 106 NIH 3T3 cells for 8 hours to allow for cell attachments. Subsequently, each cell-seeded scaffold was transferred to one station in the CSTC system and allowed to acclimatize to this environment for 36-40 hours before stretching. The scaffolds endured a 0.05N preload to establish a zero position and then was subjected to cyclic stretch elongated to a baseline stretch of 2.5%, 5%, 10%, or 15% at 0.1Hz, 0.3Hz, and 0.5 Hz for 20 minutes at 8-hour intervals for 3 days.

RNA was subsequently extracted from the cell/scaffold complexes using an RNeasy kit (Qiagen). RT-PCR was performed to synthesize cDNA with gene primers specific for murine Col I, Col III, SMA, TN-C, MMP-2, MMP-9, TGF-β1, TGF-β3 and GAPDH. PCR products were visualized on 2% agarose gel under UV light following DNA electrophoresis as shown in Figure 2.

Figure 2. Resulting RT-PCR bands for GAPDH, Col I, and Col II detected on ethidium bromide gels.

Finally, several cell/SIS-ECM scaffold complexes were fixed in 2% paraformaldehyde, permeabilized in 0.1% Triton X-100, washed with 0.5% BSA and incubated with Phallodin Alexa-488. The specimens were washed again with 0.5% BSA and stained with Hoechst dye and viewed with confocal microscopy.

All comparisons between relative expressions of each gene were performed using a Student’s t-test with significance set at a p value of 0.05.

Results
Actin staining revealed that fibroblasts responded to cyclic stretching by aligning in the direction of stretch, as shown in Figure 3.

Figure 3. 3D reconstruction of Hoechst and actin staining of NIH-3T3 fibroblasts seeded on SIS-ECM scaffolds and subjected to cyclic stretch at 0.1 Hz. Cells align in direction of applied stretch, as indicated by the white arrow.

Col I expression increased most significantly due to cyclic stretch and changes in expression were more dependent on frequency of stretch than on magnitude (Fig. 4A). Col III expression was primarily dependent on frequency of stretch but showed a decrease in expression with increasing stretch frequency. This magnitude of change in Col III expression was not as dramatic as that of Col I expression (Fig 4B).

Figure 4. (A) Col I and (B) Col III to GAPDH relative expression for NIH-3T3 cells seeded on SIS-ECM scaffold and subjected to different stretching regimens. NIH-3T3 cells cultured on TCP were used to determine basal levels of expression. # shows statistical difference from 0% stretch control and * indicates statistical difference between groups (p <0.05).

SMA expression by fibroblasts seeded on SIS-ECM scaffolds increased with mechanical stretch (Fig 5A). TN-C expression, however, decreased in response to cyclic stretching, but increased with increasing stretch frequency (Fig 5B).

Figure 5. (A) SMA and (B) TN-C to GAPDH relative expression for NIH-3T3 cells seeded on SIS-ECM scaffold and subjected to different stretching regimens. NIH-3T3 cells cultured on TCP were used to determine basal levels of expression. # shows statistical difference from 0% stretch control and * indicates statistical difference between groups (p <0.05).

MMP-2 expression decreased slightly with increasing stretch frequency. The only significant difference was detected for 0.3 Hz at 15% stretch, which showed a 50% decrease in expression compared to the no stretch condition (Fig 6A). MMP-9 expression increased with stretch frequency at 15% stretch, though all the results were not statistically different from the MMP-9 expression under the no stretch condition (Fig 6B).

Figure 6. (A) MMP-2 and (B) MMP-9 to GAPDH relative expression for NIH-3T3 cells seeded on SIS-ECM scaffold and subjected to different stretching regimens. NIH-3T3 cells cultured on TCP were used to determine basal levels of expression. # shows statistical difference from 0% stretch control and * indicates statistical difference between groups (p <0.05).

TGF-β1 expression showed dependency on frequency of stretch, increasing in a frequency-dependent manner (Fig 7A). TGF-β3 expression also significantly increased in a frequency-dependent manner at 10% and 15% stretch (Fig 7B).

Figure 7. (A) TGF-β1 and (B) TGF-β3 to GAPDH relative expression for NIH-3T3 cells seeded on SIS-ECM scaffold and subjected to different stretching regimens. NIH-3T3 cells cultured on TCP were used to determine basal levels of expression. # shows statistical difference from 0% stretch control and * indicates statistical difference between groups (p <0.05).

The analysis of the mechanical behavior of the fibroblasts seeded on the SIS-ECM scaffold showed that stiffness and maximum load decreased during each 24-hour period (ie. interval 1 to interval 3, interval 4 to interval 6) and subsequently increased after reapplying the preload to the specimen. As a result of this cyclic stretching, each specimen experienced a permanent creep (Fig 8).

Figure 8. Load-elongation curves for one fibroblast-seeded/SIS-ECM complex subjected to 10% cyclic stretch at 0.1 Hz.

Discussion
A novel CSTC system was developed and can be used to study the effects of cyclical mechanical loading on cells seeded in a particular scaffold. This system is useful for its ability to independently apply specific displacement waveforms to each scaffold while providing continuous measurement about loading capacity. In this study, the CSTC system provided further functionality by allowing the investigation of gene expression of matrix-related proteins by fibroblasts after cyclic stretching.

The results of this study indicate that the expression of Col I increased substantially due to cyclic stretching in a frequency-dependent manner, while the expression of Col III decreased. Previous studies show that in vitro Col I and Col III expression increase in response to mechanical stimuli. However, the SIS-ECM environment may allow for a more normal ratio of Col III to Col I and a normal distribution of collagen fibrils in healing tissue, explaining the improved mechanical properties in vivo.

Additionally, the increased expression of SMA, TN-C, and TGF-β1 suggest that the fibroblasts seeded in the SIS-ECM scaffold become more contractile with frequency of stretch, aligning in the direction of stretch. The increased expression of TGF-β3 may moderate contractile behavior, though the precise mechanisms are unknown since TGF-β3 has been shown to decrease contractility of cells. MMP-2 and MMP-9 expression did not change significantly in the presence of mechanical loading. This may be explained as MMP-2 and MMP-9 have been suggested to play a role in collagen degradation and remodeling, which may not be important in the mechanisms of in vivo scaffold degradation.

Critique
This study provides the foundation for the initial investigation into the in vivo healing response as affected by mechanical stimulation on SIS-ECM cell-seeded scaffolds. It devises a new CSTC system to specifically monitor the response of fibroblasts seeded on SIS-ECM scaffolds due to cyclic stretching. Furthermore, it comprehensively examines the expression of a variety of genes under various stretch conditions that may be predictive of the in vivo response.

However, this investigation contains several limitations in addressing its primary objectives. One limitation is the use of the NIH-3T3 cell line, an immortalized cell line that is not necessarily predictive of the in vivo healing response. While this cell line is useful for a preliminary study, it is more informative to look at primary cell lines or a variety of cell types since many cell populations are involved in the ECM remodeling process. Additionally, it may be useful to perform protein assays specific for the proteins of interest instead of simply gene expression, as many proteins may be post-translationally modified in unique ways that improve ECM remodeling. The study also may have benefited by including multiple durations of stretch, instead of one designated period. The authors do not mention that this interval most closely matches the in vivo environment, and thus, it is informative to find the optimum duration of stretch. Additionally, this study acknowledges that other stretching regimens should be applied in the future which may contribute to the remodeling response.

This study also did not incorporate a control scaffold (fibroblasts seeded on a collagen sponge or a silicone membrane) to measure the difference in gene expression due to mechanical loading on this scaffold compared to the SIS-ECM scaffold. The authors mention that the transmission of stretch within these control scaffold materials would be challenging. Therefore, it is beneficial to investigate other types of control scaffolds to have a valid standard of comparison to the experimental SIS-ECM scaffold. Finally, lengthening the duration of this study would also be helpful in applying these results to an in vivo environment since these SIS-ECM scaffolds typically degrade within 60-90 days.

As a side note, I would have appreciated it if some of the figures contained a scale bar to more easily allow the reader to follow their study. Overall, this study provides a good starting point for understanding the effects on cells seeded on SIS-ECM scaffolds due to mechanical loading, but further investigation is required.

Thickness sensing of hMSCs on collagen gel directs stem cell fate.

Wen Shing Leong, Chor Yong Tay, Haiyang Yu, Ang Li, Shu Cheng Wu,Duong-Hong Duc, Chwee Teck Lim, Lay Poh Tan.


Introduction:
Recently, it was found that living cells could respond to substrate elasticity. Stem cells will differentiate into specific tissue only on the substrate with similar elasticity as the native tissues, which are relative soft. But if the substrate is too soft, the stem cells will become quiescent.

Cells could also sense the thickness of the substrate. Within the interaction depth, cells would sense higher effective substrate modulus than the intrinsic modulus because of the rigid support beneath the substrate.

Among all the biological polymers, collagen is the most abundant protein in vivo; therefore it could represent ECM the best. In this study, type I collagen is used as substrate; their thickness are varied to modulate hMSCs.

Materials and Methods:
Before the experiment, the bone marrow-derived hMSCs were expanded and maintained in MSCGM. Passages 4 to 8 hMSCs were used during the experiment. They were cultured in low glucose DMEM supplemented with 10% Gold FBS, 1% L-glutamine and 1% penicillin-streptomycin. hMSCs were seeded onto thin (130μm) collagen gel, thick (1440μm) collagen gel, and collagen molecules coated coverglass in 24-wellplate for 14 days, all with a density of 1500cells/cm2. For multipotency study, adipogenic and osteogenic differentiation were induced in adipogenic and osteogenic induction medium for 14 and 21 days respectively.

To get gene expression profile, After 2 and 14 days of culture, total RNA of hMSCs was isolated for each different groups of substrates. Quantitative real time RT-PCR was performed. For immunocytochemistry, after 7 and 14 days of culture, intracellular actin filaments of the cell were stained with rhodamine conjugated phalloidin; collagen molecules were stained with anti-type I collagen, and TRITC anti-mouse IgG. For BrdU incorporation assay, after 48 hours and 14 days, cells were incubated in medium containing BrdU overnight, then stained with BrdU staining kit and cell nucleus was stained with hematocylin.

Results:
After two days of culture, the spreading area was measure for each group. It was found that projected cell area was increased with decreasing gel thickness. In addition, the hMSCs on thin collagen gel showed neurite-like extension. (Figure 1)

Fig. 1. Morphological responses of hMSCs on collagen gel of different thickness and control group at D2. (A) Spreading area of hMSCs decreased with increasing collagen gel’s thickness. (B) Phase images of hMSCs cultured on collagen gel of different thickness and control. Scale bar = 50 μm.

After 7 and 14 days, the immune-labeled cellular actin filament was visualized. It was showed that on the control (collagen molecule coated coverglass) group, the microfilaments were well defined. Actin fiber bundles were thick and aligned in parallel with the cell major axis. On thin collagen gel, actin bundles were thinner. It had similar microfilaments but the integrity and connectivity were decreased. On thick collagen gel, the actin bundle size was the smallest and most disorganized. Thick collagen gel did support the formation of long and integrated actin bundles. (Figure 2)

Fig. 2. Cytoskeleton structure of hMSCs at 7th and 14th day of culturing on thick A, D (1440μm), thin B, E (130μm) collagen gel and C, F control group. Small white dotted square at lower left corner shows enlargement of the cytoskeleton structure at highlighted area. Scale bar = 25μm.

The BrdU incorporation assay showed at the timepoints studied, none of the cells on thick gel had S-phase. Cells on thin gel demonstrated higher proliferation rate than thick gel, and the control group appeared to have to highest proliferation rate. In addition to the absence of proliferation activity, using qPCR it was showed that on thick gel the cells had downregulation in mRNA expression level of all the gene studied. (Figure 3). Non proliferation rate and downregulation of mRNA suggest that the stem cells on thick collagen gel was quiescent.

Fig. 3. Normalized mRNA expression of (A) osteogenic and (B) neurogenic specific markers of hMSCs upon seeded onto collagen gel of different thickness for 2 and 14 days. Specific upregulation of neuronal markers was observed in thin collagen gel at D14, but none of the analyzed genes was expressed in thick collagen gel over the 14 days of culturing period.

Discussion and Conclusions:
Besides the intrinsic modulus, the substrate thickness also plays an important role. Due to lack of reactive force on soft substrate, cell would have smaller spreading area and less-developed actin fiber on the softer substrate. In this study, it was showed cell spreading area and cytoskeleton development decreased with increasing collagen gel thickness. Along with non proliferative activity, it was suggest thick gel suppress gene expression and therefore the stem cell would remain quiescent state on thick gel.

Commentary:
This study studied the mechanical properties on living cells by changing the thickness of the substrate instead of changing the elastic modulus of the substrates. This is better approach because it’s easier. If the collagen concentration was changed to alter the elastic modulus, then there would be two variables in the experiment: thickness and elasticity. Since there would be variation in measure the thickness for each well, so there is no way to get exactly the same value in thickness. Therefore there is no way to determine if the change was caused by thickness effect or elasticity effect. However, in this study the authors didn’t test what is the range of interaction depth of the hMSCs. If I will perform the same experiment, I will compare more values in thickness, and a wider range of the thickness. Therefore it could be determined beyond which value the cell will not fell the difference in thickness.

Modulation of Rabbit Corneal Epithelial Cells Fate Using Embryonic Stem Cell Extract

Weijiao Zhan, Zhiping Liu, Ying Liu, Qicheng Ke, Yuanyuan Ding, Xiaoyan Lu, Zhichong Wang

Introduction

Conjunctivalization of the cornea and subsequent vision loss is an effect of corneal damage combined with limbal stem cell deficiency. Limbal stem cells provide an option for cell therapy; however, they are difficult to isolate and expand in an effective manner (can take a long time). The dedifferentiation or reprogramming of adult somatic cells provides another means to attain patient-specific stem cells for tissue regeneration. Previous studies have shown that embyronic stem cell (ESC)-derived cell-free factors and proteins are capable of reprogramming somatic cells into pluripotent cells without the use of nuclear transfer or transfection of reprogramming genes. The goal of this study was to develop a culture system to culture and dedifferentiate autologous somatic cells (rabbit corneal epithelial cells) into pluripotent cells for use in cell therapy and tissue engineering.

Methods

Cells
For this study, mouse ESC cell line ES-E14 was cultured and used for the extraction of embryonic stem cell extract. The cells were plated on 1% gelatin on tissue-treated culture plates with ESC culture media, where 50% of the media was changed every day. The ES-E14 cells were stained with anti-mouse Oct-4 antibody to confirm the undifferentiated state of the ESCs. Primary corneal epithelial cells were cultured from rabbit peripheral corneal tissue explants on tissue-treated culture plates with corneal epithelial medium.

Cell Extract Preparation
Cells were washed using PBS and cell lysis buffer, followed by centrifugation at 400x g, and resuspended with cell lysis buffer with a 40 minute incubation on ice. The cells were sonicated on ice until all cells and nuclei had been lysed. The lysate was centrifuged at 15000x g for 15 min at 4 degrees C. The supernatant was transferred to a 15 mL tube and snap-frozen and stored at -80 degrees C to remove any remaining living cells.

Cell Extract Treatment
The primary rabbit corneal epithelial cells were permeabilized using streptolysin-O (SLO) for 15 min at 37 degrees C with constant agitation. The cells were then resuspended in mES-cell extract, along with ATP, creatine phosphate, creatine kinase, and NTP, and incubated at 37 degrees C in a water bath with constant agitation. The cell membranes were resealed by transfer to epithelial cell culture media with CaCl2 and seeded onto a tissue culture plate for 2 hours at 37 degrees with 5% CO2. The media was then replaced with complete epithelial cell culture media and changed every other day. Colonies were isolated and transferred to ESC medium for culture.

Histology and Immunohistochemistry
This was performed after 24-48 hours of culture for Oct-4, SSEA1, K3/K12, p63, ABCG2, and Vimentin. They were counterstained with Hoechst 33342, followed by imaging using a confocal laser scanning microscope. The cells were also tested for gene expression using RT-PCR, stained with BCIP/NBT Phosphate Substrate System to test for AKP activity, and tested for tumorigenicity (teratoma formation after subcutaneous injection).

Results
The results indicated mES-colony like structures in passage 5 or week 3 of culture. The colonies were maintained in ESC medium for at least 14 weeks in culture, but began to flatten out. Oct-4 expression was detected in P2, and peaked at P9 (week 4), decreasing in subsequent weeks. SSEA1 was detected in P9, but not in P18, and neither of these markers were detected in the control samples. K3 and p63, which are specific for corneal tissue and progenitor cells, were also detected in the cells, indicating that the cells were not completely reprogrammed to and ES state, but were still capable of returning to the start of their lineage. Vimentin was not detected, indicating a lack of fibroblast contamination. ESC extract induced cells formed teratomas and were capable of differentiating into all three germ layers. AKP staining revealed positive staining at P9, but weaker staining at P18

Figure 1

Representative phase-contrast micrographs of cells. A-C: SLO+ corneal epithelial cell extract-induced cells (p-Pc) P1 (day 8), P3 (day 12), P5 (day 14); D-F: cells without any treatment (b-Pc) P1 (day 3), P3 (day 9), P5 (day 14); G-I: SLO+ES cell extract-induced cells (e-Pc) P1 (day 8), P3 (day 12), P5 (day 14); J-L: e-Pc P9 (wk 4), P12(wk 6), P18 (wk 8). The magnification of panel E is 50×, the other panels are at 100×. The three groups maintained similar morphology at P1 (day 8; A, D, and G). Some fibroblast-like cells took place in p-Pc group (B and C). The P3 cells in b-Pc control exhibited aging appearance with vacuoles and larger size, and could not survive through P5 (E and F). At P3 (day 14) of e-Pc, among the aging epithelial cells, colonies with small cells and vague boundaries showed up (H). At P5 (week 3), colonies that closely resembled to mES colonies could be readily identified by their morphology (I). This phenomenon was maintained for at least 14 weeks in culture, corresponding to 26 passages, but the colonies gradually became flat (J-L).

Figure 2

mRNA expression of Oct-4, K3, and p63 with GAPDH as an internal control for P2 in all groups, E₁₄ and P6, P9, P18 of e-Pc. After the mES cell extract treatment, Oct-4 mRNA was detected in P2 (day 12), reached its peak at P9 (week 4), and decreased in later passages. It remained undetectable in the two control groups. Expression of corneal tissue-specific marker K3 mRNA increased as passage in experiment group, and progenitor cell marker p63 was also found in these cells.

Figure 3

Expression of pluripotency-associated proteins Oct-4 and SSEA1 in e-Pc with immunofluorescent staining. The scale bar represents 50 μm. Oct-4 and SSEA1 proteins were found in P9 (week 4), not in P18 (week 8) cells.

Figure 4

Expression of corneal-epithelium-related proteins in different conditions and passages with immunofluorescent staining. A-C: p63, ABCG2, and K3 (green, positive cells; blue, nuclei) for P2 in all groups and P9, P18 of e-Pc. D: Vimentin for P6 in p-Pc group. E: Vimentin for P9 in e-Pc group. The scale bar in B represents 50 μm. Corneal tissue-specific marker K3 and progenitor cell markers, p63 or/and ABCG2 were still found in different passages of e-Pc. Vimentin, an intermediate filament protein and a characteristic of keratocytes and fibroblasts was not detected in P9 cells of e-Pc. But it was positive in P6 of p-Pc.

Figure 5

Teratoma formation examination and alkaline phosphatase (AKP) staining. A: HE staining shows teratoma from ES cells and P9 (wk 4) e-Pc containing multiple tissues, including epithelium (E), neural (N), muscle (M), and glandular structures (G). The scale bar represents 50 μm. B: AKP staining in P9 (wk 4) and P18 (wk 8) e-Pc (magnification, 100×). AKP staining was positive at P9 (week 4), and positive but weaker at P18 (week 8). Small and cohesive colonies were mostly observed in P9 cells. Flatter, larger and more migratory colonies were noted in P18 cells.


Critique
The paper shows that embyronic stem cell extract is capable of dedifferentiating rabbit corneal epithelial cells; however, the cells are incapable of maintaining this state and can only be partially reprogrammed to multipotency. It was also demonstrated they can be quickly dedifferentiated, expanded, and maintained for many weeks. Although this study has demonstrated that embryonic cell extract, the study did not demonstrate that there is global expression of precursor and embryonic markers. Also, there was no study done to demonstrate that these partially dedifferentiated cells were capable of achieving similar functionality as rabbit embryonic stem cells and corneal epithelial cells. The paper was pretty thorough in testing both qualitatively through immunostaining as well as quantitatively through RT-PCR, and is a good first step; however, these cells need to be compared to other cells (ES cells, progenitor cells, corneal epithelial) to determine their functionality. After this functionality has been determined, in vivo studies regarding recovery of vision loss and prevention of conjunctivalization of the cornea need to be performed.

Directed differentiation of Human induced pluripotent stem cells generates active motor Neurons

Introduction:

iPS cells may provide a means of generating functional motor neurons for regenerative medicine. Many diseases, such as amyotrophic lateral sclerosis and spinal muscular atrophy, result in the loss of motor neurons. Previous studies have shown the ability of human embryonic stem cells to generate motor neurons. Hence, these methods, when applied to iPS cells, yield motor neurons that are electrophysiologically functional and seem to follow a normal development. This study suggests that iPS cells could be used in regenerative medicine to repair the functional loss of motor neurons and could also provide a means of modeling the developmental progression of motor neuron diseases in vitro.

Methods:

Control: A well studied hESC line – HSF1

Two different protocols were used to generate functional mature motor neurons from iPS cells.

Embryoid body differentiation protocol used EBs cultured on an hESC media lacking FGF2 for 1 week and were treated with retinoic acid for one week. The EBs were also treated with a Sonic Hedgehog pathway agonist. This resulted in the neuralization of the embryoid bodies, indicated by the expression of neural progenitor markers such as Brn2, Sox3, and Pax6. Progressive differentiation of the pluripotent cells to neural progenitors was observed, though only a certain fraction of the HSF1 and iPS cells showed neural markers detected via immunostaining.

An adherent approach was used to generate neural rosettes from HSF1 and multiple iPS cell lines (iPS1, iPS2 and iPS18). The rosettes were mechanically isolated and plated on to laminin coated dishes along with Retinoic acid and Shh for 1 week. Neurotrophic factors such as BDNF, CNTF and GDNF were added and Shh concentration was lowered by 75% to allow the cells to differentiate for 3-5 weeks. Transition of the cells from being nestin-positive neural progenitors to mature motor neurons was detected via immunostaining.

The differentiation protocols were used to show that once specified to a neuronal fate, the hESCs and the iPS cells were comparable in generating motor neurons.

Antibody immunostaining was performed to detect neural progenitor markers and fluorescence and DIC images were collected. Whole cell, patch clamp analysis was also performed for electrophysiological activity of the differentiated neurons.

Results:

With regards to the differentiation efficiencies of the HSF1 cell like and the iPS cell line, the authors reported that there were significant differences between the two. They suggested that the discrepancy was due to the variability between different cell lines, which might have attributed to the inconsistency in efficiency. However, the authors went on to say that among those EBs who showed a preference to neural fate (with the expression of Brn2, Sox3, and Pax6), the number of differentiated cells were similar (Fig. 1). Upon further treatment of the cells with retinoic acid, Shh agonists and neurotrophic factors, the EBs were cryosectioned and stained for motor neuron progenitor markers Nkx6.1 and Olig2 (Fig 2).

Figure 1: hESC cells and iPS cells derived from EB protocol stained for neural progenitor markers Brn2, Sox3 and Pax6.

Figure 2: HSF1 and hiPS2 cells stained for neural progenitor markers as well as mature motor neuron markers (Nxk6 and Olig2). Percentages for Sox3+ cells: ~59.1% HSF1and ~57.6% of hiPS2 cells.

Cells obtained from the adherant approach were stained for neural progenitor markers (nestin) as well as for mature motor neuron markers (BIII-tubulin, chat and Islet1+). Similar percentages of Islet1+ cells were reported for the HSF1 and hiPS2 cells were reported, indicating that the differentiation efficiency was similar between the two cell lines. These cells were also stained for a Hb9 activity (a transcription factor expressed in mature motor neutons). The authors used Hb9-driven green fluorescent protein reporter gene to identify activity. Hoxa5 gene staining shows that the cells expressed a rostral cervical character. (Fig.3)

Figure 3: HSF1 cells and hiPS2 cells were stained for neural progenitor markers (nestin) as well as for mature motor neuron markers (BIII-tubulin, chat and Islet1+) (3A-H). HSF1, hiPS1 and hiPS18 derived motor neurons were stained for ChAT activity and Hoxa5 as markers for spinal cord neurons.

It is a characteristic of motor neurons to fire repetitive action potentials as a response to current injection. Whole cell patch clamping was used to asses the excitability of the neurons. Roughly half of the Hb9:GFP+ neurons (derived from iPScs and hESCs) responded with repetitive action potentials. The cells also showed choline acetyltransferase activity which is typically seen in motor neurons as a response to electrical stimulation. (Fig 4). These results demonstrate the potential of iPS cells to generate active motor neurons.

Figure 4: Electrophysiological properties of HSF1 and hiPS2 cells that stained positive for ChAT and expressed Hb9-GFP reporter gene.

Critique:

Though this paper shows the capacity of iPS cells to become motor neurons, there are several inconsistencies in their results. Multiple iPSC clones were used for these experiments, but the date often showed only one cell line or another. They did not mention any data on the behavior of physiological neurons as a positive control, despite recurrently placing emphasis on the use of iPSC-derived motor neurons for regenerative medicine. Most of this study was exclusively qualitative, without any focus on the extent of gene expression in the derived neurons. Additionally, the electrophysiological studies did not use the same amount of current to test motor neurons derived from different cell lines. The frequency of action potentials also varied between cell lines. Further quantitative experiments and better controls should be done to better understand the behavior of iPSC derived motor neurons.

Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins

Dohoon Kim, Chun-Hyung Kim, Jung-Il Moon, Young-Gie Chung, Mi-Yoon Chang,

Baek-Soo Han, Sanghyeok K, Eungi Yang, Kwang Yul Cha, Robert Lanza, and Kwang-Soo Kim

Introduction

Currently, all methods to derive induced pluripotent stem cells include risks associated with the use of viruses and mutation(s) during DNA transduction. However in this study, authors generated iPS cells from human fibroblasts by delivering the four reprogramming factors (Oct4, Sox2,Klf4, and c-Myc) with a cell penetrating peptide (CPP). The resulting cells maintained pluripotency for more than 35 passages and differentiated into all three germ layers both in vitro and teratomas.

The goal of the study was to generate pluripotency without viral and DNA vectors in order to make protein-induced human iPS (p-hiPS) cells suitable for clinical trials and patient-specific cells for regenerative medicine.

Procedure

To overcome the major obstacle for the delivery of proteins, which is their limited ability to cross the cellular membrane, authors hypothesized that the cell penetrating peptides can be used. Cell penetrating peptides can cross the membrane because they contain high amounts of basic amino acids such as lysine and arginine. To test the hypothesis, red fluorescent protein (RFP) was fused with a 9 arginine CPP and exposed to COS7 and human newborn fibroblasts (HNFs). Within a few hours,it was shown that the fluorescent protein was delivered into the cell (Fig. S1).

Figure S-1

Then, the four human reprogramming proteins were fused to a 9 arginine-CPP for HEK 293 cell lines, which resulted in high expression of the four proteins using Western blotting analyses (Fig. S2).

Figure S-2

16 hours treatment with the four proteins with 6 days incubation in ES Media resulted in iPS-like colonies with positive alkaline phosphatase (AP) activity after the 6th cycle. The overall process to generate iPS-like colonies took 8 weeks which is twice the amount of time required for virus transduction reprogramming process, and the efficiency of the process was 0.001% compared with 0.01% of the virus transduction.

Then to test for pluripotency, the expression of ES markers such as AP, Oct4, Nanog, tumor-rejection antigen (TRA)1-60, stagespecific embryonic antigen (SSEA) 3 and SSEA 4 was quantified using qRT-PCR. The expression pattern was identical to the embryonic stem cells. Furthermore, bisulfite sequencing analyses indicated that the promoter of the pluripotency

genes Nanog and Oct4 were significantly demethylated compared with the densely methylated parental HNF cells'.

At last, the p-hiPS cells formed embryoid bodies (EBs) by suspension culture and differentiated into cells of all three germ layers (Fig. 2d).

Figure 2d

Also, after transplantation of p-hiPS cells under the kidney capsule of nude mice for 6 to

8 weeks, teratoma formation was observed. The teratomas included neural tissues
(ectoderm), epidermal tissues (ectoderm), striated muscle (mesoderm), adipose tissue
(mesoderm), cartilage (mesoderm), respiratory epithelium (endoderm), and intestinal-like
epithelial tissues (endoderm) (Fig. 2e).

Figure 2e

Prolidase-dependent regulation of TGF c and TGF β receptor expressions in human skin fibroblasts

Arkadiusz Surazynski, Wojciech Miltyk, Izabela Prokop, Jerzy Palka

All figures and captions are from the above authors.

Introduction

An important part of many physiological processes is ECM degradation. For processes such as wound healing or angiogenesis, the ECM needs to be remodeled to make room for cell proliferation to occur. An important regulator of ECM is TGF-β. It stimulates the production of collagen and protease inhibitors that prevent ECM breakdown.

On the other hand, prolidase is an enzyme that catalyzes the breakdown of ECM and releases proline (Pro) and hydoxyproline (HyPro) from ECM collagen. Past studies have shown that it is also involved in the regulation of collagen biosynthesis at a transcriptional level and even VEGF and HIF-1α production, which makes it an important enzyme for processes such as wound healing or angiogenesis.

In this paper, they tried to determine whether prolidase is also involved in the regulation of TGF-β.

Methods

In this experiment, they used human skin fibroblast cells and they extracted the proteins from the cells via sonication and centrifugation. The prolidase was exposed to gly-proline and trichloroacetic acid was used to stop the prolidase reaction. The amount of proline released was determined by adding Chinard’s reagent, measuring the absorbance of the solution at 515 nm and calculating the results according to proline standards.

Western blotting was also used to measure protein activity. The gel was transferred onto a nitrocellulose membrane and stained with antibodies specific for: phospho-MAPK, phospho-AKT, phospho-mTOR, TGF-β1 receptor, TGF-β1, TGF-β3 and β-actin. Secondary antibodies were used as well to illuminate the results.

Results

The results showed that when enzyme inhibitors, Cbz-Pro and PEP, were added, prolidase activity decreased in a dose-dependent manner. However, TGF-β1 expression was also decreased by the inhibitors.

Fig. 1. Prolidase activity in confluent human skin fibroblasts (Control) and the cells treated with different concentration of Cbz-Pro or PEP for 24 h in medium containing 0.1% FBS. Mean values from six independent experiments ±S.D. are presented. * = P less than 0.05, **= P less than 0.001

In order to test whether prolidase activity is affecting TGF-β1 expression, they tried adding the products of prolidase activity, such as Pro and HyPro, to the solution to see if it would affect TGF-β1. The results showed that adding these products can indeed counteract the effects of the inhibitors of TGF-β1 and even induce TGF-β1 expression if enough is added. Thus, TGF-β1 expression is dose-dependent on prolidase products.

Figure 2. Western Blot of TGF-β1 in a medium of confluent human skin fibroblast. In (A), cells are treated with enzyme inhibitors and in (B) cells are treated with different amounts of proline and hydroxyproline.

As for the expression of TGF-β1 receptors, they found that the enzyme inhibitors also decreased the production of the receptors, while Pro and HyPro increased production in a dose-dependent manner.

Figure 3. Western Blot of TGF-β1 receptors in a medium of confluent human skin fibroblast. In (A), cells are treated with enzyme inhibitors and in (B) cells are treated with different amounts of proline and hydroxyproline.

Activated TGF-β1 receptors activate MAPK, AKT and mTOR via phosphorylation. While the prolidase inhibitors had no effect of MAPK, they decreased the expression of phospho-AKT and phosphor-mTOR. Furthermore, adding Pro and HyPro had no effect on MAPK, but increased expression of phosphor-AKT and phosphor-mTOR in a dose-dependent manner.

Figure 4. Western Blot of Phospho-AKT in a medium of confluent human skin fibroblast. In (A), cells are treated with enzyme inhibitors and in (B) cells are treated with different amounts of proline and hydroxyproline.

Figure 5. Western Blot of Phospho-mTOR in a medium of confluent human skin fibroblast. In (A), cells are treated with enzyme inhibitors and in (B) cells are treated with different amounts of proline and hydroxyproline.

Interestingly, the mTOR inhibitor, rapamycin, was found to also decrease prolidase activity in a dose-dependent manner. Thus, the results suggest that Pro and HyPro are modulators for mTOR activation and rapamycin inhibits mTOR activation by decreasing prolidase activity. This relationship would allow mTOR to detect the availability of Pro before it initiates factors to promote collagen synthesis.

Figure 6. Prolidase activity in confluent human skin fibroblast with varying concentration of Rapamycin.

Discussion

While TGF c was mentioned in the title, I did not see it anywhere else in the paper.

Also, they noted that TGF-β3 was not affected in the same conditions, but it was not clear whether they meant that the prolidase products had no effect or if even the enzyme inhibitors had no effect on TGF-β3 expression. The data for TGF-β3 was not shown so it is impossible to tell. Also, no hypothesis was provided as to the difference in results between TGF-β1 and TGF-β3. Thus we don’t know for sure whether this is due to some error in the experimental procedures or a different reason, such as its structural properties.

Finally, they mentioned how prolidase inhibitors decrease the expression of TGF-β1 receptors and signals activated by TGF-β1. However, it is unclear whether the decrease in the expression of the signals is due to the decrease in the number of receptors, which would lead to the activation of fewer signals, or if the prolidase inhibitors act directly on the expression of the signals.

Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA

Luigi Warren, Philip D. Manos, Tim Ahfeldt, Yuin-Han Loh, Hu Li, Frank Lau, Wataru Ebina, Pankaj K. Mandal, Zachary D. Smith, Alexander Meissner, George Q. Daley, Andrew S. Brack, James J. Collins, Chad Cowan, Thorsten M. Schlaeger, Derrick J. Rossi

All figures and captions are from the above authors.

Introduction

Induced pluripotent stem cells (iPSCs) allow researchers to examine the processes of development and differentiation, and can provide a source of autologous cells for the treatment of diseases. Current approaches to induced pluripotency, which include enforced gene expression via retroviral or excisable vectors and serial protein transduction, result in low efficiencies. Furthermore, the modification of cellular genome leads to risks of recombination or mutation, and recombinant proteins for transduction are difficult to generate and isolate. This paper examines an alternative to the current methods by inducing pluripotency through the administration of synthetic mRNA modified to overcome innate antiviral responses.

Summary

To develop modified RNAs, the researchers complexed RNA with a cationic vehicle to facilitate cellular uptake by endocytosis. Immunogenic responses were evident with dose-dependent toxicity after transfecting murine embryonic fibroblasts and human epidermal keratinocytes with synthetic RNA coding for GFP. The synthetic RNA was treated with phosphate to deactivate protein kinase R, a global repressor of protein translation. Modified nucleoside bases were shown to improve cell viability by reducing interferon signaling (Figure 1A-D). Furthermore, media supplementation with B18R, a virus decoy receptor for type I interferons, increased cell viability. Due to RNA and protein degradation, expression is transient and repetitive transfections are required to maintain high levels of protein expression over an extended period of time. After transfecting modified RNA with myogenic transcription factor (MYOD) into murine C3H10T1/2 cells, myotubes emerged that stained positive for myogenic factors (Figure 1I).

The authors repeatedly transfected modified RNA with Yamanka factors OCT4, KLF4, SOX2, and c-MYC into human ESC-derived fibroblasts. Cells were cultured in low oxygen conditions with a KMOS stoichiometry to promote conversion to iPSCs. Human ESC-like colonies emerged toward the second week of transfection and were mechnically picked after 20 days, leading to 14 prospective iPSC lines (Figure 2C). The authors also transfected somatic derived cells with modified RNA encoding LIN28. Daily transfection led to numerous hESC like colonies in all cell lines which expressed pluripotency markers OCT4, NANOG, TRA-1-60, TRA-1-81, SSEA3, and SSEA4 (Figure 2D).

To determine the effectiveness of RNA reprogramming, cells were evaluated by RT-PCR. All cells had robust expression of OCT4, SOX2, NANOG, and hTERT (Figure 3A). RNA-induced pluripotent stem cells (RiPSCs) had molecular signatures very similar to those of human ESC and highly divergent from parental fibroblasts (Figure 3C). Unsupervised hierarchical clustering analysis revealed that RiPSCs clustered more closely to hESC than did virally derived iPSCs (Figure 3D). In embryoid bodies generated from five RiPSC derivations, cardiomyocytes were observed in the majority. RiPSCs were capable of neuron and endodermal cell differentiation (Figure 4B) along with in vivo teratoma formation (Figure 4C).

The efficiency of reprogramming with RNA was measured according to the expression of pluripotency markers TRA-1-60 and TRA-1-81. In one particular experiment, RNA induced conversion efficiency was found to be two times higher than virus-based derivations. Efficiency was markedly improved when a low-oxygen condition was combined with a five-factor cocktail with RNA encoding LIN28. The authors tested a protocol which directly compared transfection of either KMOS-modified RNAs or KMOS retroviruses in dH1f fibroblasts. Whereas RNA transfected cultures were overgrown with ESC-like colonies by the 16^th day, colonies did appear on retroviral cultures until day 24. Furthermore, iPSC derivation efficiency was found to be 36 fold higher with modified RNA (Figures 5E, F). The authors successfully transfected RiPSCs with modified RNA encoding MYOD resulting in differentiation to myogenic lineage (Figure 6).

Commentary:

The research presented in this paper appears to be a significant step forward for the field of induced pluripotency. The authors were able to effectively demonstrate that RNA technology is highly efficient in comparison with current reprogramming techniques while eliminating the risk of genomic modification and mutation. They followed a thorough protocol for ensuring pluripotency of the cells by testing for various markers, gene expression, and developmental potential. While the authors tested methods of reducing immune response to RNA, they did not mention which was the most effective. The upregulation of interferon genes which was still evident after RNA modification needs to be addressed in future studies to determine long-term safety. The authors could have strengthened the paper by testing the differentiation of RiPSCs into various cell types. Overall, the work done in this paper has strong support for the use of RiPSCs in the future, but more studies need to be conducted to determine its application and safety.

VEGF Induces Differentiation of Functional Endothelium From Human Embryonic Stem Cells. Implications for Tissue Engineering

Marilyn B. Nourse, Daniel E. Halpin, Marta Scatena, Derek J. Mortisen, Nathaniel L.

Tulloch, Kip D. Hauch, Beverly Torok-Storb, Buddy D. Ratner, Lil Pabon and Charles E. Murry

Introduction:

Human embryonic stem cells (hESCs) have shown promise in the application for regenerating tissues with specific cell types. Currently however, the obstacles that arise relate to the inefficiency of the current techniques used to promote and distinguish the functional endothelial cells from differentiating hESCs. This article attempts to use a vascular endothelial grown factor (VEGF) as treatment and demonstrated a 4-5 fold enrichment of endothelial cells which in turn were implanted to form vessels and deemed viable in vivo. This implication will facilitate future endeavors involving tissue-engineered implants.

Methods:

Undifferentiated hESCs were allowed to turned into embryoid bodies (EBs) by separated confluent cultures into small clumps. VEGF-induced and control EBs, human umbilical vein endothelial cells (HUVECs), were then dispersed into single cells and stained with fluorescent conjugated antibodies (CD31, VECad, vWF, CD45[negative control]). The EBs were grown for 4 or 14 days and then monolayers of cells (HUVECs and DC31) were serum starved and then stimulated with TNFa. The RNA collected was quantified via RT-PCR and the protein analysis was taken via Western blotting. The cells were also assayed for tubule formation and colony forming ability (methylcellulose assay). Three million CD31 cells were mixed into a gel/culture medium under standard tissue culture conditions. The porous scaffolds were then prepared with hESC-influenced endothelial cells seeded. The scaffolds were later implanted into rats and processed for histology after ten days. The fluorescent images were lastly attained by confocal microscopy.

Results/Discussion:

Figure 1 portrays the induction of endothelium with VEGF. The test of cell differentiation showed that the treatment with VEGF resulted in more protein than standard cultures.

An increase of the dosage of VEGF also correlated to a positive expression until around 50ng/mL.

Figure 2 illustrates the immunofluorescence of the VEFG-induced EBs with the various markers. The plots for day 10 and 14 was to justify that while all the other markers coexpressed, CD45 never was detected in all conditions and time variables. CD45 is a marker for hematopoetic stem cells which indicate unwanted differentiation.

Figure 3 shows that although VEGF induces the differentiation in favor of endothelium it does not effect the proliferation in EBs.

Figure 4 further confirmed the cells' morphology and also that they were negative for CD45 and positive for the markers CD31, VeCAD, and vWF which is what was anticipated.

VEGF was shown to have a 4.7-fold increase in the number of differentiating endothelial cells but as for proliferation analysis, it resulted in no difference.

Figure 5 show that in vivo hESC-derived endothelial develop into robust vascular networks. These are different magnifications of CD31 stained A,C,D and unstained B in different magnifications.

Discussion/Applications:

Overall, this article discovered a novel and important finding of how to induce differentiation for functional endothelium. This could potentially help with the process of differentiation of mesodermal precursor cells that could expand the cell populations in hESC cultures and create a useful source for cardiac tissue engineering applications. Also ECs derived from hESC may improve neovascularization in transplant and scaffolds for tissue engineering. if combined with collagen gels in vivo, they could help organize into vascular networks as well.

The only critique for this article is that they do not indicate whether or not the endothelial cells achieve sustained expansion and stability of vascular cells. Vascular commitment is essential in making sure that the grafts, transplants, or other engineering products are sustainable in potential patients.