The Raclure Method
Spontaneously differentiated cells often appear in hESC cultures in the center or at the edges of the colonies (Fig. 1A). These cells are mechanically eliminated from the culture before each passage because they tend to promote the differentiation of the undifferentiated cells remaining in the dish. We observed that these scraped cells (“raclures” in French) remain multipotent since after replating they can differentiate into multiple cell types, including neurons and cardio-myocytes, depending on the culture conditions (data not shown). We report here a procedure to differentiate these raclures into MSCs.
Figure Figure 1.. Phase-contrast microscopy. (A): Undifferentiated (left) and differentiated (right) human embryonic stem cell colonies (original magnification ×10×4). The darker area in the center and the left part of the center colony contain differentiated cells that are mechanically eliminated before each passage. These scraps, or raclures, contain pluripotent cells. (B, C): Morphology of the mesenchymal cells at confluence (original magnification ×10×4) (B) and 3 weeks after confluence (original magnification ×40×4) (C). Both cultures exhibit typical fingerprint-like patterns. P37R forms bi- or trilayer stratified epithelium, whereas P51R generally forms monolayers. Abbreviation: HEPM, human embryonic palatal mesenchymal.
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To obtain MSCs, raclures are first plated in D10 medium (DMEM + 10% FBS + 1% penicillin/streptomycin + 1% nonessential amino acids) until a thick, multilayer epithelium of cells develops. This requires at least 4 weeks, with weekly medium changes. Once the epithelium is formed, it can survive in culture for many months with minimal medium change (once every 3 weeks). The MSCs are then isolated by dissociation of this epithelium using a mixture of trypsin, collagenase type IV, and dispase for 4–6 hours and replating of the cells in D10 medium. Two or 3 days after the passage, the population of cells obtained is morphologically uniform, and cells exhibit a characteristic regular fingerprint-like morphology when they are observed at confluence by phase-contrast microscopy (Fig. 1B). The resulting cultures can then be passaged at least 20 times.
Since the hESCs were cultured on MEFs, it was important to determine whether MEFs constituted a significant proportion of the raclures. To assess the percentage of irradiated MEFs that were scraped with the raclures, fluorescence-activated cell sorting analysis was performed on the day of the scraping and 1 week later using TRA 1-85, a human-specific antibody, to differentiate the MEFs from the human cells. This analysis revealed that about 5% of the raclures consisted of cells of murine origin and that a week after plating the raclures, more than 99.5% of the cells were human (data not shown). As discussed below, in the established MSC cultures, no cells of mouse origin could be detected, suggesting that the MEFs present in the raclures were completely mitotically inactivated by the irradiation.
The procedure to isolate MSCs is reproducible, since 15 independent experiments yielded similar populations of cells. The dissociation of the epithelium could be performed from 50 to more than 180 days after initial plating of the raclures without noticeable differences in the type of cells obtained. We report here on the detailed characterization of two of these isolates, P37R and P51R.
P37R was produced by dissociation of the epithelium 180 days after plating of raclures of H1 cells at passage 37. P51R was produced by dissociation of the epithelium 50 days after plating of raclures of H1 cells at passage 51. The P37R cells are small and spindle-shaped (Fig. 1C). The P51R cells are larger (Fig. 1C). P51R is strictly contact inhibited and forms a monolayer. P37R cells are also contact inhibited but form a bilayered or trilayered epithelium. The morphology of the other isolates varied between these two extremes.
To determine the growth characteristics of the P37R and P51R isolates, cultures at passage 5 were trypsinized every 3 days, counted, and replated at 10,000 cells per ml. As shown in Figure 2A, the growth rate of the cells started to decrease at passage 17, and the cultures reached senescence between passage 25 and passage 30.
Figure Figure 2.. Growth characteristic and karyotypes of the P37R and P51R isolates. Plots illustrating the growth rate and population doubling time of the P37R and P51R isolates and the HEPM control. Abbreviation: HEPM, human embryonic palatal mesenchymal.
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Spectral karyotype analysis of the P37R and P51R isolates at passage 13 revealed no abnormalities, suggesting that the karyotype of these cells is stable (data not shown).
As a first step toward the characterization of the P37R cells, we performed preliminary expression analysis using a custom-made cDNA glass slide micro-array produced by the Albert Einstein College of Medicine Micro-Array Facility. These arrays contain 6,000 human cDNAs. Two comparisons were performed. The first comparison was between MSC-P37R cells and undifferentiated hESCs. The second was between the MSC-P37R cells and mesenchymal cells termed human embryonic palatal mesenchymal (HEPM) that we obtained from the American Type Culture Collection (Manassas, VA, http://www.atcc.org) . More than 40% of the spots on the array were upregulated or downregulated at least 1.5-fold when the MSC-P37R cells were compared with undifferentiated hESCs. By contrast, this number was only 12% when the MSC-P37R were compared with the HEPM cells, demonstrating that MSC-P37R resemble HEPM cells much more than undifferentiated hESCs.
We then examined the most highly expressed genes in the MSC-P37R cells as compared with the hESCs and found that out of the 25 most highly expressed genes in the MSC-P37R, 15 had been identified previously in a serial analysis of gene expression analysis [28, 29] as genes that were highly expressed in bone marrow and cord blood mesenchymal cells, suggesting that the hESC-derived cells had a profile of expression similar to primary MSCs. Table 1 illustrates genes in the hESC-derived MSC-P37R cells that are the most highly induced compared with hESCs.
Table Table 1.. Most highly expressed genes in P37R as compared to hESCs
Flow Cytometry Analysis
MSCs isolated by different methods present a somewhat variable profile of antigen expression and share many characteristics with endothelial, epithelial, and muscle cells, complicating the task of finding a simple identifying marker. Nevertheless, there is a general consensus that MSCs are negative for CD45 and CD34 but positive for markers such as SH2, SH3, and SH4 [1, 17]. To characterize the surface antigen profiles of P37R and P51R, we used the above-mentioned antibodies plus others that have been reported to be expressed on only some MSC populations. As controls, we used the HEPM cell line. The results of this analysis are shown in Figure 3. P37R, P51R, and HEPM were positively stained for CD44, CD71 (transferrin receptor), CD73 (SH3), CD105 (endoglin, SH2), CD166, and HLA-ABC and negative for CD34 and CD45 (Fig. 3). Staining for CD13 revealed a very weak expression of this epitope for P37R and P51R as compared with HEPM. Interestingly, staining for SSEA-4 , an antigen expressed on hESCs and in early human embryos, was high for P37R and P51R but negative for HEPM. The human-specific antibody TRA 1-85  was used to exclude the possibility that the cells were not of human origin. The immunophenotypes of P37R and P51R were therefore compatible with the hypothesis that these cells were MSCs. To determine whether there was any contamination by cells of murine origin that could originate from MEFs that would have survived the irradiation, we also tested the MSCs with TRA 1-85, a human-specific antibody. No cells of mouse origin could be detected (Fig. 3).
Figure Figure 3.. Surface antigen expression of the P37R and P51R isolates. Cells at or near confluences were trypsinized and stained with fluorochrome-conjugated antibodies against CD13, CD34, CD44, CD45, CD71, CD73, CD105, CD166, HLA-ABC, SSEA-4, and TRA 1-85. Dead cells were gated out using propidium iodide. The table on the right summarizes the results. The white overlay represents cells stained with the isotype control antibodies. Abbreviations: FITC, fluorescein isothiocyanate; HEPM, human embryonic palatal mesenchymal; HLA, human leukocyte antigen; MSC, mesenchymal stem cell; PI, propidium iodide; SSEA, stage-specific embryonic antigen.
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Functional Differentiation of P37R and P51R
To confirm that P37R and P51R were MSCs, we performed functional differentiation assays. We focused on osteogenesis and adipogenesis since MSCs derived from multiple organs have the capacity to differentiate along these pathways,
Osteogenic differentiation was performed using the β-glycerophosphate method as previously described for primary adult MSCs . After 3 weeks of differentiation, a majority of the cells had differentiated into osteoblasts, as demonstrated by calcium deposition in the matrix visualized with alizarin red staining (Fig. 4A). HEPM cells were used as a positive control .
Figure Figure 4.. Functional differentiation. (A): Phase-contrast micrographs of HEPM, P37R, and P51R after osteogenic differentiation and staining with Alizarin red. (B): Phase-contrast micrographs of HEPM, P37R, and P51R after adipogenic differentiation using either the IBMX or the SWH method and staining with oil red O. Original magnification ×10×20 (main panel), ×10×40 (inset). Abbreviations: HEPM, human embryonic palatal mesenchymal; IBMX, 3-isobutyl-1-methylxanthine; SWH, serum withdrawal/hypoxia.
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Adipogenic differentiation was performed by two different methods. At first, attempts were made by the classic 3-isobutyl-1-methylxanthine (IBMX) method [17, 21, 33]. This resulted in adipocytic differentiation, but the lipid vesicles obtained were small (Fig. 4B). Much larger vesicles were obtained using an alternate method of adipogenic differentiation that we developed in the laboratory.
This new method relies on the serendipitous observation that P37R cells grown in serum-free medium (KSR + DMEM/F-12 + l-glutamine + NEAA) accumulates small cytoplasmic vesicles that are lightly stained by oil red O, on the finding that hypoxia enhances lipid accumulation , and on the report that FGF enhances PPAR-γ ligand-induced adipogenesis of MSC . As seen in Figure 4B, incubation of MSCs in these conditions resulted in the differentiation of most of the culture into cells that contain large cytoplasmic vesicles that are brightly stained with oil red O. Another advantage of this new serum withdrawal/hypoxia (SWH) method is that it is much less sensitive to initial plating density (data not shown) .
To characterize the adipocytes produced from hESC-derived MSCs, we performed a real-time RT-PCR analysis on RNA extracted from MSC-P37R and MSC-P51R either undifferentiated or differentiated using the IBMX and SWH methods. As controls, we also tested undifferentiated hESCs, purified adult hematopoietic cells (CD45+), human primary breast adipocytes, and HEPM cells. Primers specific for transcription factors involved in adipogenesis (PPAR-γ2 and SREBf1c), for proteins involved in lipid droplets (perilipin and adipophilin) and lipid metabolism (lipoprotein lipase and GAPDH), or for cytokines produced by adipocytes (adiponectin, PGAR, and leptin) were used as described by Fink et al. .
This analysis revealed that both methods induce the expression of adipocytic markers but that there were differences between the two differentiation protocols (Fig. 5).
Figure Figure 5.. Quantitative real-time reverse transcription polymerase chain reaction (PCR) analysis of adipocytes derived from P37R and P51R. Total RNAs were extracted from CD45-positive peripheral blood monocytes, from undifferentiated hESCs (H1 passage 35), and from HEPM, P37R, and P51R cells before or after adipocytic differentiation using either the IBMX or the SWH method. Quantitative real-time PCR analysis was performed using primers specific for SREBF, PGAR, PPARγ2, ADPF, GAPDH, perilipin, LPL, ACDC, LEP, and β2-microglobulin (B2M) on a Roche LightCycler. Histograms illustrating expression of the tested genes relative to expression of B2M in each cell type normalized to the same ratio in the CD45-positive cells (Materials and Methods). The results show that both methods induce adipocyte-specific genes, but to different extents (see text). Abbreviations: ACDC, adiponectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEPM, human embryonic palatal mesenchymal; IBMX, 3-isobutyl-1-methylxanthine; LPL, lipoprotein lipase; MSC, mesenchymal stem cell; PGAR, proliferator-activated receptor γ-induced angiopoietin-related; PPR, peroxisome proliferator-activated receptor; SWH, serum withdrawal/hypoxia.
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In the case of MSC-P37R, PPAR-γ2, a highly specific adipocyte marker, was expressed at relatively high levels (5% of fully mature adipocytes) prior to differentiation. The SWH method led to further induction of PPAR-γ2 to approximately 15% of the level in mature adipocytes. The SWH protocol also led to a large increase in the level of perilipin, led to a moderate increase in adipophilin production, and had little or no effect on SREBf1c expression. By contrast, the IBMX method led to a small decrease in PPAR-γ2 expression but to dramatic increases in SREBf1c, adipophilin, and PGAR expression, three genes that are expressed at high levels in adipocytes. Neither treatment induced the production of adiponectin, leptin, or lipoprotein lipase. Results with MSC-P51R were similar except that the induction of PPAR-γ2, perilipin, and adipophilin were not as strong and that trace amounts of lipoprotein lipase could be detected.
We conclude from the oil red O stain and from these expression data that both the P37R and the P51R isolates have adipocytic potential, that the SWH method is more efficient than the IBMX method for the induction of adipocytic differentiation, and that the two methods lead to slightly different types of adipocytes or to adipocytes at different stage of maturation.
hESC-Derived MSCs Support the Growth of Undifferentiated hESCs
Recent reports have shown that bone marrow-derived MSCs can support the growth of hESCs  and hematopoietic stem cells (HSCs) . To test whether hESC-derived MSCs can support the growth of hESCs, undifferentiated H1 cells were passaged on either irradiated P51R, irradiated P37R, or irradiated MEFs, and the resulting H1 cells were compared by a variety of assays. After 32 successive passages, the H1 cells had similar undifferentiated morphologies regardless of the feeder used (Fig. 6A, 6B). Immunostaining with SSEA-4  antibodies (Fig. 6C–6H) and real-time RT-PCR analysis for Oct-4 (Fig. 6I) revealed that the level of expression of these markers, which are known to be expressed at high levels in H1 cells, was high regardless of the feeder used.
Figure Figure 6.. P37R and P51R isolates can serve as feeder for hESCs. (A, B): Micrographs (original magnification ×10×4) showing that hES cells grown on MEFs (A) or on P51R for 32 weeks (B) have similar morphology. (C–H): Micrographs (original magnification ×10×10) of hESC colonies grown on P37R (C, D), P51R (E, F), or MEFs (G, H); fixed; stained with a mouse anti-human stage-specific embryonic antigen-4 antibody; and revealed by fluorescein isothiocyanate-labeled rat anti-mouse IgG. All colonies are strongly stained. (I): Quantitative real-time reverse transcription-polymerase chain reaction analysis demonstrating high-level expression of Oct-4 in hESCs grown on P37R or P51R feeders compared with Oct-4 expression in the feeders alone. (J): hESCs grown on P51R were cocultured with FH-B-hTERT to induce hematopoietic differentiation. The dotplots on the top (obtained after 14 days of coculture with FH-B-hTERT) demonstrate that the number of CD34-positive cells obtained is similar to that obtained using hESCs grown on MEFs. The histograms on the bottom illustrate that hESCs grown on P51R and cocultured on FH-B-hTERT for 13 or 17 days yield erythroid and myeloid colonies after plating in methylcellulose. (Each bar is the average of two separate experiments.) These results show that hESCs grown on P37R and P51R retain their capacity to differentiate into hematopoietic cells. Abbreviations: BFU-E, burst forming unit-erythroid; CFU-E, colony forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte erythroid macrophage megakaryocyte; CFU-GM, colony forming unit-granulocyte-macrophage; CFU-M, colony forming unit-macrophage; FSC, forward scatter; hES, human embryonic stem; hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast.
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To functionally characterize H1 cells grown on P37R and P51R, we differentiated them into hematopoietic cells by coculture with FH-B-hTERT (a human fetal hepatocyte cell line) cells as described in Qiu et al. . After 2 weeks of coculture, the percentage of CD34-positive cells was determined by flow cytometry. These experiments revealed that similar numbers of CD34-positive cells were produced whether MEF, P37R, or P51R were used as feeders (Fig. 6J). Methylcellulose assays confirmed and extended these results since they revealed that hESCs grown on the P51R feeders yielded erythroid and myeloid colonies in relatively large numbers (Fig. 6J; ). Together, these data show that H1 cells grown on P37R and P51R retained their morphology, the expression of hESC markers, and their ability to differentiate into hematopoietic cells. These data suggest that these feeders are able to support the growth of undifferentiated H1 cells that can be differentiated in hematopoietic cells. Additional studies will be required to determine whether hESCs grown on P37R and P51R retain their ability to differentiate into other cell types.