Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach

<div><p>Background</p><p>A number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach.</p><p>Methodology/Principal Findings</p><p>Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both <i>in vitro</i> and <i>in vivo</i>.</p><p>Conclusions/Significance</p><p>These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture.</p></div

<i>In Vitro</i> differentiation potential of protein iNPCs.

<p>(A) Human protein iNPC-derived neural cells exhibited typical neuronal morphology. (B) Quantification of MAP2⁺ neurons, GFAP⁺ astrocytes and O4⁺ oligodendrocytes among total cells. Data are represented as mean ± SD. (C) Differentiation potential of iNSCs into neurons by TUJ1, astrocytes as determined by GFAP, and oligodendrocytes by O4 antibody via immunocytochemistry. DAPI staining is shown in blue. (D) MAP2⁺ neurons derived from iNPCs. (E) Dotted Synapsin expression in proximity of the TUJ1⁺ nerve fibers suggested morphological synapse formation. (F) The major population of iNPC-derived neurons was GABA⁺. (G) A small population of neurons were TH⁺. TUJ: β-Tubulin class III; MAP2: Microtubule-associated protein 2; TH: Tyrosine hydroxylase.</p

Assessment of TAT recombinant protein transduction.

<p>(A) Visualization of fusion proteins in in suspension cultured HFFs transduced with 10 μg/ml TAT-EGFP for 24 h. Scale bars represent 200 μM. (B) To determine protein stability, 10 μg/ml of TAT-EGFP proteins were added to HFFs for 24, 48 and 72 h, and the presence of transduced protein in the cells were analyzed by flow cytometery (M; monolayer and S; 3D sphere culture). (C) The uptake and stability of the TAT fusion protein were visualized by TAT immunostaining of transduced human fibroblasts after 24 and 48 h post-TAT-SOX2 transduction (Scale bars: 200 μm). (D) The middle panels represent double staining TAT and SOX2 with higher magnification in transduced cells at 24 h post-transduction (50 μm). The nuclei were counterstained with DAPI. (E) Determining the time required for protein transduction. Relative expression of neural markers (<i>SOX2</i> and <i>PAX6</i>) induced by 10 μg/ml TAT-SOX2 with a 48 h transduction interval (d0: Non-transduced cells).</p

<i>In Vivo</i> transplantation of iNPCs.

<p>(A) Transplanted GFP⁺ protein iNPCs were TUJ⁺ at 10 days post-transplantation into the brain of a rat pup. Nuclei were counterstained with DAPI. (B) GFAP⁺ astrocytes were derived from GFP⁺ iNPCs <i>in vivo</i>.</p

Characterization of established protein iNPCs.

<p>(A) Phase contrast of established iNSCs morphology at higher passages (p20). (B) Real-time analysis of iNPC and ES-derived NPC (ES-NPC) for neural progenitor markers. (C) Expressions of Nestin, PAX6 and SOX2 were visualized by immunostaining. Nuclei were counterstained with DAPI. (D) Flow cytometric analysis of neural progenitor markers (PAX6 and SOX2) and proliferation marker Ki67 in iNPCs. (E) Single-cell-per-well experiments illustrated that the established protein iNPC grew clonally. (F) Real-time analysis of secondary spheres of iNPCs for neural progenitor markers. (G) Relative expression levels of region-specific marker genes over human fibroblasts, whose expression was considered to be 1 for all genes.</p

Optimization of the effect of rISL1 protein on hESCs.

<p>(A) To evaluate the effect of discontinuous (2 h/day) or continuous rISL1 protein addition on hESCs (Royan H5) differentiation, cells were treated continuously or discontinuously from days 1–8 post initiation of differentiation. <i>Isl1</i> qRT-PCR analysis of differentiated cells at day 8 showed higher significant endogenous <i>Isl1</i> expression in hESCs in the continuous protocol.Thus continuous treatment was applied in the next steps. )* : P<0.05((B) To determine the best concentration of rISL1 protein for cardiac differentiation, cells were treated with four different concentrations of recombinant protein: 10, 20, 30, and 40 µg/ml in continuous treatment of hESCs during days 1–8 after initiation of differentiation. During differentiation, cells that were treated by 10 and 20 µg/ml rISL1 protein were morphologically similar to hematopoietic and endothelial progenitors, while the 30 and 40 µg/ml concentrations showed cardiomyocyte and muscular appearances. It seems that 30 and 40 µg/ml rISL1 protein are better concentrations for cardiac differentiation. )* : P<0.05((C) qRT-PCR analysis of differentiated cells at day 8 by different concentrations of rISL1 also showed that 40 µg/ml of the rISL1 protein induced more endogenous <i>Isl1</i>, but less <i>Mef2c</i> and <i>Nkx2.5</i> expressions. )* : P<0.05((D) Schematic diagram of the differentiation protocol by the addition of rISL1 protein (40 µg/ml), which was added after induction with Activin A (days 1–8). qRT-PCR analysis of endogenous <i>Isl1</i> expression in hESCs demonstrated that treated cells expressed higher significant endogenous <i>Isl1</i> than the untreated control. )* : P<0.05((E) The percentage of beating clusters in continuous treatment of hESCs by 40 µg/ml rISL1 protein during days 1–8 after differentiation initiation in comparison with the control (vehicle-treated) group. The percentage of beating clusters in the rISL1-treated group was significantly higher than the untreated group at day 14 after plating (75±10% vs. 20±2.5%). )* : P<0.05((F) rISL1 treatment resulted in a 3.2±0.5 fold increase in the number of beating areas in comparison with untreated control group. rISL1 also caused a 2.2±0.4 fold increase in the other hESC line, Royan H6, which shows the reproducibility of this protocol for another hESC line. )* : P<0.05((G) In order to assess the expression of cardiac-specific genes, we collected samples at 3 stages: day 3 after plating (the day of rISL1 removal); day 14 after plating (day of maximum beating); and day 20 after plating (day that beating decreased and cells were mature) by qRT-PCR in two hESC lines. Target genes were normalized by the reference gene <i>Gapdh</i>. The relative expression was calculated by dividing the normalized target gene expression of treated hESCs with rISL1 protein and elution buffer (as control) with that of the undifferentiated state (day 0). All data are statistically significant in comparison with undifferentiated state (day 0) otherwise marked with “ns” (ns: P>0.05). a: P<0.05 in comparison with control group (elution buffer treated group). All data were represented as log2-linear plots.</p

ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells

<div><h3>Background</h3><p>Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes.</p> <h3>Methods</h3><p>We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1) recombinant protein into the cells.</p> <h3>Results</h3><p>We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1–8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4) doubled. This protocol was also reproducible for another hESC line.</p> <h3>Conclusions</h3><p>This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.</p> </div

Daily qRT-PCR analysis in aggregate differentiation of hESCs.

<p>Undifferentiated aggregates of hESCs were treated by Activin A for 1 day and then for 4 days by BMP4. At day 5, the aggregates were plated without cytokines. The data show the maximum expression of the mesoendodermal marker, <i>Brachyury</i>, one day after Activin A treatment (day 2 after differentiation initiation). By continuing differentiation with BMP4 for the next 4 days <i>Isl1</i>, a marker of precardiac mesoderm, and <i>Actinin</i> were reached to their highest expression level. <i>Isl1</i> expression was remained at high level for the next 3 days and by decreasing its expression, <i>Mef2c</i>, a cardiac progenitor marker showed its maximum expression and after that, other cardiac progenitor genes, <i>Gata4 Nkx2.5</i> and <i>Tbx5</i> reached to their highest expression level respectively. Finally, the expression of <i>MHC</i> and <i>cTnT</i>, which are structural cardiomyocytes markers, got to maximum level (Fig. 1). These data shows that 3-dimentional structures of the cells are very important for cardiac differentiation and aggregated differentiation method enhances cardiac differentiation and functionality.Target genes were normalized by the reference gene <i>Gapdh</i>. The relative expression was calculated by dividing the normalized target gene expression of the treated sample with that of the undifferentiated state (day 0). All data represented as log2-linear plots. All data are statistically significant otherwise marked with “ns” (P>0.05).</p