Differentiation of Human Embryonic Stem Cells ... - Stem Cells Journals

EMBRYONIC STEM CELLS Differentiation of Human Embryonic Stem Cells into Bipotent Mesenchymal Stem Cells EMMANUEL N. OLIVIER,a ANNE C. RYBICKI,b ERIC E. BOUHASSIRAa a

Einstein Center for Human Embryonic Stem Cell Research, Department of Medicine, Division of Hematology and Department of Cell Biology, bDepartment of Medicine, Division of Hematology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, USA Key Words. Differentiation • Adipocytes • Osteocytes • Mesenchymal stem cells • Human embryonic stem cells

ABSTRACT Mesenchymal stem cells (MSCs) are multipotent progenitors that can be found in many connective tissues, including fat, bone, cartilage, and muscle. We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs that does not require the use of any feeder layer. The cells obtained with this procedure are morphologically similar to bone marrow MSCs, are contactinhibited, can be grown in culture for about 20 to 25 passages, have an immunophenotype similar to bone marrow MSCs (negative for CD34 and CD45 and positive for CD13,

CD44, CD71, CD73, CD105, CD166, human leukocyte antigen [HLA]-ABC, and stage-specific embryonic antigen [SSEA]-4), can differentiate into osteocytes and adipocytes, and can be used as feeder cells to support the growth of undifferentiated hESCs. The ability to produce MSCs from hESCs should prove useful to produce large amounts of genetically identical and genetically modifiable MSCs that can be used to study the biology of MSCs and for therapeutic applications. STEM CELLS 2006;24: 1914 –1922

INTRODUCTION

methods and found only subtle differences between the different preparations [6]. In addition to bone marrow, MSCs have been isolated from multiple organs, including synovial fluid, cartilage, deciduous teeth, cord blood, amniotic fluid, placenta, and adipose tissue [7–14]. Recently, MSCs have also been obtained by differentiation of hESCs [15]. Since no unique marker of MSCs has been identified, investigators have relied on a series of functional and morphological criteria to identify them. These criteria include growth on plastic, resistance to trypsin, presence of specific cell surface antigens, and potential to differentiate into adipocytes, chondrocytes, and osteocytes [16 –18]. We report here a novel method to reproducibly differentiate H1 (WA01) [19] human embryonic stem cells (hESCs) into MSCs.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can be found in many connective tissues including fat, bone, cartilage and muscle. Because of this differentiation potential, MSCs are generally considered to have a large therapeutic potential, particularly in the areas of cell therapy and regenerative and reconstructive medicine [1–5]. MSCs were first recognized as distinct cell populations in the 1990s and were isolated by a variety of procedures. Cell preparations with similar morphology and properties have been termed bone marrow stromal cells, mesenchymal stem cells, skeletal stem cells, or multipotent adult progenitor cells by different investigators. Although these different names may designate cells with different differentiation potential, for the sake of simplicity, we will use the term mesenchymal stem cells in a generic manner in this report. MSCs are very rare cells that were first isolated from the bone marrow. The classic method to isolate MSCs from bone marrow relies on their capacity to adhere to plastic and their resistance to trypsinization during passage in culture in simple culture medium (i.e., Dulbecco’s modified Eagle’s medium [DMEM] plus 10% fetal calf serum). Lodie et al. have characterized human bone marrow MSCs prepared by four different

MATERIALS

AND

METHODS

Culture of hESCs

The H1 hESCs were cultured on ␥-irradiated (80 Gray) feeder cells (mouse embryonic fibroblasts [MEFs] or hES cell-derived MSCs [ES-MSCs]) plated at 75,000 cells per cm2 at 37°C and 5% O2 and 7.5% CO2. hESCs medium contained DMEM/ Ham’s F-12, 20% Knockout Serum Replacer (KSR), 2 mM L-glutamine, minimal essential medium nonessential amino acid

Correspondence: Eric Bouhassira, Ph.D., Department of Medicine, Division of Hematology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. Telephone: 718-430-2188; Fax: 718-824-3153; e-mail: bouhassi@aecom. yu.edu Received December 23, 2005; accepted for publication April 17, 2006; first published online in STEM CELLS EXPRESS April 27, 2006. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0648

STEM CELLS 2006;24:1914 –1922 www.StemCells.com

Olivier, Rybicki, Bouhassira solution (NEAA), 0.1 mM penicillin-streptomycin 1% (all from Gibco, Grand Island, NY, http://www.invitrogen.com), 4 ng/ml basic fibroblast growth factor l (R&D Systems Inc., Minneapolis, http://www.rndsystems.com; or ProSpect-Tany, Technogene, Rehovot, Israel, http://www.prospec.co.il) and 0.1 mM 1-thioglycerol (Sigma-Aldrich, St. Louis, http://www. sigmaaldrich.com). The culture medium was changed daily, and the cells were passaged once a week.

Expression Profiling Microarray design, RNA extraction, probe labeling, and data processing and analysis were as described in Rybicki et al. [20].

Fluorescence-Activated Cell Sorting Analysis Cells were harvested using 0.05% trypsin-0.53 mM EDTA (Gibco) and after neutralization resuspended in staining buffer (Dulbecco’s phosphate-buffered saline [DPBS] ⫹ 5% KSR) at a concentration of 106 cells per ml. For each antibody used, 105 cells were stained. Antibodies for CD13, CD71, CD105, human leukocyte antigen (HLA)-ABC, isotype control for IgG1, IgG2a, IgG3, and fluorescein isothiocyanate (FITC) rat anti-mouse IgG(H⫹L) were from eBioscience; CD34 FITC, CD45PE, CD73PE, isotype control IgG1⌲ FITC, IgG1⌲ PE, and FITC rat anti-mouse IgG1 were from BD Biosciences (San Diego, http://www.bdbiosciences.com); CD44, SSEA-4, and TRA 1-85 were from the Developmental Studies Hybridoma Bank (Iowa City, IA, http://www.uiowa.edu/⬃dshbwww). At least 10,000 events were acquired for each sample using a FACSCalibur (BD Biosciences). Dead cells were gated out using propidium iodide staining (1 ␮g/ml).

Functional Differentiation of ES-MSCs To induce osteogenic differentiation, cells were seeded at 3,000 cells per cm2 in D10 medium (DMEM with 10% fetal bovine serum [FBS]). At 50%–70% confluency, growth medium was supplemented with 100 nM dexamethasone (Decadron; Merck & Co., Whitehouse Station, NY, http://www.merck.com), 50 ␮M ascorbic acid-2-phosphate, and 10 mM ␤-glycerophosphate (all from Sigma-Aldrich). The medium was replaced every 3– 4 days for 21 days. Cultures were washed twice with phosphatebuffered saline (PBS), fixed in a solution of ice-cold 70% ethanol for 1 hour, and stained for 10 minutes with 1 ml of 40 mM Alizarin red (Sigma-Aldrich) [21]. For adipogenic differentiation, cells were seeded at 104 cells per cm2, and two methods were used. In the first, at confluence, cells were put in D10 medium supplemented with 1 ␮M dexamethasone, 0.2 mM indomethacin, 10 ␮g/ml insulin, and 0.5 mM 3-isobutyl-1-methyl-xanthine (all from Sigma-Aldrich). In the second, at confluence, cells were put in hES medium in partial hypoxia (5% O2). In both cases, medium was replaced every 3– 4 days for 21 days. Cells were washed three times with PBS, fixed in 10% formalin for 1–2 hours, and stained for 15 minutes with fresh oil red O solution (Sigma-Aldrich) [22].

Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis Total RNAs were isolated using RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) and treated with DNase I (Roche Molecular Biochemicals, Manheim, Gerwww.StemCells.com

1915

many, http://www.roche-applied-science.com). Total RNA isolated from normal human adipose tissue (a gift from Dr. M. Hawkins) was used as a positive control for adipocyte-specific gene expression. Reverse transcription was performed with Superscript III first strand kit (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with approximately 5 ␮g of RNA and random decamer primers. For each sample, 20-␮l reactions were set up in duplicate, each with 10 ␮l of Quantitect SYBR Green PCR Kit (Qiagen), 0.5 pmol of each primer, and 2 ␮l of cDNA (equivalent of 50 ng of RNA). In the case of Oct-4 detection, detection was performed using the single-tube Quantitect SYBR Green reverse transcription-polymerase chain reaction (RT-PCR) kit (Qiagen). The following primers were used (gene symbol is given in square brackets).

␤2-Microglobulin [B2M]: forward: 5⬘-agcaaggactggtcttt-3⬘, reverse: 5⬘-ctcgatcccacttaactatct-3⬘. ADD1/SREBP1c [SREBF1]: forward: 5⬘-aggatctggtggtggg-3⬘, reverse: 5⬘-ggttctcctgcttgagt-3⬘. Peroxisome proliferator-activated receptor (PPAR)-␥ 2 [PPARG2]: forward: 5⬘-cttgcagtggggatgt-3⬘, reverse: 5⬘-ctttggtcagcgggaa-3⬘. Perilipin [PLIN]: forward: 5⬘-tccacccagttcacagc-3⬘, reverse: 5⬘-agatggtgtccttcagc-3⬘. aP2 [FABP4]: forward: 5⬘-atttgacgaagtcactgc-3⬘, reverse: 5⬘-catcctctcgttttctctttat-3⬘. Lipoprotein lipase [LPL]: forward: 5⬘-tgctcgtgctgactct-3⬘, reverse: 5⬘-cgggaatgaggtggcaa-3⬘. Adiponectin [ACDC]: forward: 5⬘-ttccgggaatccaagg-3⬘, reverse: 5⬘-ggtaaagcgaatgggc-3⬘. Peroxisome proliferator-activated receptor ␥-induced angiopoietin-related gene (PGAR) [ANGPTL4]: forward: 5⬘-ctccgccatttttggt-3⬘, reverse: 5⬘-gccttgtaggcttccc-3⬘. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GAPD]: forward: 5⬘-ctataaattgagcccgcag-3⬘, reverse: 5⬘-aaatccgttgactccga-3⬘. Adipophilin [ADPF] as published by Fink et al. [23]: forward: 5⬘-cgctgtcactggggcaaaaga-3⬘, reverse: 5⬘-atccgactccccaagactgtgtta-3⬘. leptin [LEP] as published by Fink [23]: forward: 5⬘-ggagtgacggtcccacactg-3⬘, reverse: 5⬘-ccatgtaataaacccagagtgtgatc-3⬘. To control for the amount of cDNA in each reaction, ␤2M was used as an internal standard. The amplifications were performed on a Roche LightCycler using three-temperature cycling consisting of denaturation step at 95°C for 8 seconds, an annealing step at 58°C for 18 seconds, and an extension step at 72°C for 20 seconds. At the end of each reaction, melting curves were acquired during cooling from 95°C to 65°C. Levels of expression were normalized to the level of B2M expression in each cells and to the level of expression in the CD45-positive cell controls using the following formula:

Human ES Cells into Mesenchymal Stem Cells

1916

Immunostaining with SSEA-4 Antibody hESC colonies were fixed in situ with formalin for 15 minutes. After three rinses with PBS, cells were incubated overnight at 4°C with mouse anti-human SSEA-4 antibody (DHSB) or isotype control (eBioscience, San Diego, http:// www.ebioscience.com) (approximately 2 ␮g per 106 cells) in PBS ⫹ 5% KSR. Antibody localization was performed using rat anti-mouse IgG (H⫹L) immunoglobulin conjugated to fluorescein isothiocyanate (eBioscience). Differentiation of hESCs into hematopoietic cells was performed as previously described [25]. Briefly, hESCs were cocultured with fetal hepatocytes (FH-B-hTERT) [26] in DMEM ⫹ 20% FBS ⫹ 1% penicillin/streptomycin ⫹ 1% nonessential amino acids. Half of the medium was replaced every 3 days. After 2 weeks, cells were harvested and plated on methylcellulose. Colonies were counted 17 days later.

RESULTS The Raclure Method

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.

2exponent (–(Ct[tested gene in tested cells] – Ct[B2M in tested cells])/ (Ct[tested gene in CD45 cells]– Ct[B2M in CD45 cells]), where Ct is the cycle threshold at which PCR products were first detected.

Karyotype Analysis Spectral karyotyping was performed as described before [24].

Growth Curves Population doubling time was calculated following the formula (logN/log2)/t, where N is the number of cells at confluence divided by the initial number of cells, and t is the number of hours in culture. The growth curve was established by multiplying the initial number of cells by the amplification fold for each passage.

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. 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

Olivier, Rybicki, Bouhassira

1917

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).

RNA Profiling 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 MSCP37R 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) [27]. 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.

Flow Cytometry Analysis 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.

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. www.StemCells.com

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 [30], 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 [31] 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).

1918


Table 1. Most highly expressed genes in P37R as compared to hESCs Fold overexpressiona

Gene description Matrix Gla protein Fragile X mental retardation, autosomal homolog 1 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) Collagen, type VI, ␣3 Insulin-like growth factor binding protein 3 Protein tyrosine phosphatase, receptor type, C Osteoblast-specific factor 2 (fasciclin I-like) GRO1 oncogene (melanoma growth-stimulating activity ␣) Fibronectin 1 Insulin-like growth factor 2 (somatomedin A) GRO1 oncogene (melanoma growth-stimulating activity ␣) NGFI-A binding protein 1 (ERG1 binding protein 1) Ubiquitin fusion degradation 1-like Keratin 5 Cysteine knot superfamily 1, BMP antagonist 1 Zinc finger protein homologous to Zfp92 in mouse Aquaporin 1 (channel-forming integral protein, 28 kD) Collagen, type III, ␣1 (Ehlers-Danlos syndrome type IV, autosomal dominant) Lysyl oxidase Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (lysine hydroxylase) 2 Aspartate ␤-hydroxylase Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), ␣ polypeptide II Human mRNA for MTG8a protein, complete cds S100 calcium-binding protein A9 (calgranulin B) Hexabrachion (tenascin C, cytotactin) Aspartate ␤-hydroxylase Cadherin 11, OB-cadherin (osteoblast) Collagen, type IV, ␣2 Antigen identified by monoclonal antibodies 12E7, F21, and O13 Latent transforming growth factor ␤ binding protein 2 Cytoplasmic linker 2

98.1 68.0 46.0 43.1 42.5 40.1 38.8 36.2 35.8 34.6 34.4 33.4 31.8 31.0 29.7 28.0 26.3 25.0 24.6 23.0 20.9 19.8 19.6 17.6 17.3 17.1 16.7 16.6 16.5 16.4 16.1

a

P37R expression/hESC expression. Abbreviations: BMP, bone morphogenetic protein; hESC, human embryonic stem cell.

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 ␤-glycerophosphate method as previously described for primary adult MSCs [21]. 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 [32]. Adipogenic differentiation was performed by two different methods. At first, attempts were made by the classic 3-isobutyl1-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 [34], and on the report that FGF enhances PPAR-␥ ligand-induced adipogenesis of MSC [35]. 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) [36]. 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-␥2 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. [23]. This analysis revealed that both methods induce the expression of adipocytic markers but that there were differences between the two differentiation protocols (Fig. 5). In the case of MSC-P37R, PPAR-␥2, 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-␥2 to approximately 15% of the level in mature adipocytes. The SWH protocol also


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.

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-␥2 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-␥2, 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. www.StemCells.com

1919

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.

hESC-Derived MSCs Support the Growth of Undifferentiated hESCs Recent reports have shown that bone marrow-derived MSCs can support the growth of hESCs [37] and hematopoietic stem cells (HSCs) [38]. 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 [19] antibodies (Fig. 6C– 6H) and realtime 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.

1920


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, PPAR␥2, ADPF, GAPDH, perilipin, LPL, ACDC, LEP, and ␤2-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 ␥-induced angiopoietinrelated; PPR, peroxisome proliferator-activated receptor; SWH, serum withdrawal/hypoxia.

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. [25]. After 2 weeks of coculture, the percentage of CD34-positive cells was determined by flow cytometry. These experiments revealed that similar numbers of CD34positive 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; [19]). 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.

DISCUSSION We describe here a simple method to derive bipotent MSCs from hESCs. The cells obtained grow very robustly, have a stable karyotype, are contact inhibited, senesce after about 20 passages, have an immunophenotype similar to that of MSCs derived from other sources, have adipocytic and osteocytic potential, and can support the growth of hESCs and hematopoietic progenitors.

The cells that we obtained appear similar to the hESCderived MSCs described recently by the Studer laboratory [15], but our method has the advantage of not requiring any feeder layer of animal origin that could complicate the use of these cells for clinical purposes. Whether the bipotency of the P37R and P51R isolates reflects a true bipotency of individual cells or the presence of two types of progenitor cells in the isolates remains unproven since we have not yet characterized clonal population of cells. However, microscopic observations showing an almost complete differentiation into adipocytes favor the hypothesis that the P37R and P51R cells are truly bipotent. Derivation of mesenchymal cells from human ES cells should help us understand the specification events that occur during early human development and could have useful clinical applications, since these cells have a large therapeutic potential, particularly in the areas of cell therapy and regenerative and reconstructive medicine. From an experimental point of view, derivation of MSCs from hESCs has specific advantages over derivation from other sources. In the first place, a virtually unlimited amount of genetically identical MSCs can be produced from hESCs. Second, the ability to genetically manipulate the hESCs before the production of the MSCs should provide unparalleled experimental opportunities to study the molecular basis of the totipotency of the MSCs cells, as well as pathways that lead to human osteogenic and adipocytic differentiation. Finally, the ability of


1921

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 transcriptionpolymerase 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; CFUGM, 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.

the hESC-derived MSCs to support the growth of hESCs is also of practical importance [39, 40] since they represent an almost unlimited source of autogenic feeder that eliminates the risks of transmission of xenogeneic pathogens and since they can be conveniently cultured for more than 20 passages, allowing the production of large amounts of cells.

ACKNOWLEDGMENTS E.E.B. is supported by NIH Grants R01-DK56845, P01HL55435, and P20-GM075037. A.C.R. is supported by NIH Grants HL-68962 and HL-38655. The monoclonal antibodies for CD44, SSEA-4, and TRA1-85 (developed by J.T. August and J.E.K. Hildreth, D. Solter, and P.W. Andrews, respectively)

REFERENCES 1

Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004;95:9 –20.

2

Zhao RC, Liao L, Han Q. Mechanisms of and perspectives on the mesenchymal stem cell in immunotherapy. J Lab Clin Med 2004;143: 284 –291.

www.StemCells.com

were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA. We thank Dr. C. Montagna of the Albert Einstein College Of Medicine Genome Imaging Facility for the spectral karyotyping analyses, Dr. Meredith Hawkins and Dr. Wei Jie for the generous gift of RNA from human primary adipocytes, and Dr. Philip Scherer for useful discussions.

DISCLOSURES The authors indicate no potential conflicts of interest.

3

Liechty KW, MacKenzie TC, Shaaban AF et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282–1286.

4

Lee JW, Kim YH, Kim SH et al. Chondrogenic differentiation of mesenchymal stem cells and its clinical applications. Yonsei Med J 2004; 45(suppl):41– 47.

1922


5

Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: Characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004;8:301–316.

6

Lodie TA, Blickarz C, Devarakonda TJ et al. Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction. Tissue Eng 2002;8:739 –751.

25 Qiu C, Hanson E, Olivier E et al. Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development. Exp Hematol 2005;33:1450 –1458.

7

Jones EA, English A, Henshaw K et al. Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis. Arthritis Rheum 2004; 50:817– 827.

26 Wege H, Le HT, Chui MS et al. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology 2003;124:432– 444.

8

Alsalameh S, Amin R, Gemba T et al. Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage. Arthritis Rheum 2004;50:1522–1532.

9

Fukuchi Y, Nakajima H, Sugiyama D et al. Human placenta-derived cells have mesenchymal stem/progenitor cell potential. STEM CELLS 2004; 22:649 – 658.

10 Gang EJ, Hong SH, Jeong JA et al. In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells. Biochem Biophys Res Commun 2004;321:102–108. 11 Gang EJ, Jeong JA, Hong SH et al. Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. STEM CELLS 2004;22:617– 624. 12 Tsai MS, Lee JL, Chang YJ et al. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod 2004;19:1450 –1456. 13 Yen BL, Huang HI, Chien CC et al. Isolation of multipotent cells from human term placenta. STEM CELLS 2005;23:3–9. 14 Rodriguez AM, Elabd C, Amri EZ et al. The human adipose tissue is a source of multipotent stem cells. Biochimie 2005;87:125–128.

24 Schrock E, du Manoir S, Veldman T et al. Multicolor spectral karyotyping of human chromosomes. Science 1996;273:494 – 497.

27 Yoneda TPR. Mesenchymal cells from the human embryonic palate are highly responsive to epidermal growth factor. Science 2005;213: 563–565. 28 Tremain N, Korkko J, Ibberson D et al. MicroSAGE analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages. STEM CELLS 2001;19:408 – 418. 29 Silva WA Jr., Covas DT, Panepucci RA et al. The profile of gene expression of human marrow mesenchymal stem cells. STEM CELLS 2003;21:661– 669. 30 Kannagi R, Cochran NA, Ishigami F et al. Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J 1983;2: 2355–2361. 31 Williams BP, Daniels GL, Pym B et al. Biochemical and genetic analysis of the OKa blood group antigen. Immunogenetics 1988;27:322–329. 32 Facer SR, Zaharias RS, Andracki ME et al. Rotary culture enhances pre-osteoblast aggregation and mineralization. J Dent Res 2005;84: 542–547.

15 Barberi T, Willis LM, Socci ND et al. Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med 2005;2:e161.

33 Sekiya I, Larson BL, Vuoristo JT et al. Adipogenic differentiation of human adult stem cells from bone marrow stroma (MSCs). J Bone Miner Res 2004;19:256 –264.

16 Colter DC, Class R, DiGirolamo CM et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A 2000;97:3213–3218.

34 Wada Y, Sugiyama A, Yamamoto T et al. Lipid accumulation in smooth muscle cells under LDL loading is independent of LDL receptor pathway and enhanced by hypoxic conditions. Arterioscler Thromb Vasc Biol 2002;22:1712–1719.

17 Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147. 18 Jones EA, Kinsey SE, English A et al. Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum 2002;46:3349 –3360. 19 Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.

35 Neubauer M, Fischbach C, Bauer-Kreisel P et al. Basic fibroblast growth factor enhances PPARgamma ligand-induced adipogenesis of mesenchymal stem cells. FEBS Lett 2004;577:277–283. 36 Lee RH, Kim B, Choi I et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004;14:311–324.

20 Rybicki AC, Fabry ME, Does MD et al. Differential gene expression in the kidney of sickle cell transgenic mice: Upregulated genes. Blood Cells Mol Dis 2003;31:370 –380.

37 Cheng L, Hammond H, Ye Z et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.

21 Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A 2001;98:7841–7845.

38 Kobune M, Kawano Y, Ito Y et al. Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells. Exp Hematol 2003;31:715–722.

22 Kasturi R, Joshi VC. Hormonal regulation of stearoyl coenzyme A desaturase activity and lipogenesis during adipose conversion of 3T3–L1 cells. J Biol Chem 1982;257:12224 –12230.

39 Xu C, Jiang J, Sottile V et al. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. STEM CELLS 2004;22:972–980.

23 Fink T, Abildtrup L, Fogd K et al. Induction of adipocyte-like phenotype in human mesenchymal stem cells by hypoxia. STEM CELLS 2004;22: 1346 –1355.

40 Stojkovic P, Lako M, Stewart R et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. STEM CELLS 2005;23:306 –314.