Mesenchymal differentiation of hESCs (lines H1 [WA-01] and H9 [WA-09]) [9
] was induced by plating undifferentiated hESCs on a monolayer of murine OP9 stromal cells [10
], in the presence of 20% heat-inactivated FBS in alpha MEM medium. OP9 cells have been previously shown to induce blood cell differentiation from mouse ES cells [11
]. After 40 d of coculture, cells were harvested and sorted by FACS for CD73, a surface marker expressed in adult MSCs [5
] (A). An average of 5% CD73+ cells was obtained from the mixed culture of OP9 and differentiated hESC progeny. CD73+ cells were replated in the absence of stromal feeders on tissue culture plates and expanded in alpha MEM medium with 20% FBS for 7–14 d. We next established the membrane antigen profile of the resulting population of flat spindle-like cells. The H1- and H9-derived CD73+ cells expressed a comprehensive set of markers that are considered to define adult MSCs, including CD105(SH2), STRO-1, VCAM (CD106), CD29(integrin β1), CD44, ICAM -1(CD54), ALCAM(CD166), vimentin, and alpha smooth muscle actin (B and C). The cells were negative for hematopoietic markers such as CD34, CD45, and CD14. They were also negative for neuroectodermal, epithelial, and muscle cell markers including nestin, pancytokeratin, and desmin (data not shown). The human identity of these presumed mesenchymal cells (termed hESMPC-H1.1, -H1.2, -H1.3, -H1.4, and -H9.1) was confirmed for all experiments by immunocytochemistry for human nuclear antigen to rule out the possibility of contamination with OP9 cells (Figure S1
Isolation and Characterization of hESMPCs
To further characterize hESMPCs, we performed genome-wide expression analysis using oligonucleotide arrays (Affymetrix U133A). The expression profiles of hESMPC-H1.2 and hESMPC-H9.1 were compared with that of human primary adult MSCs. Housekeeping genes for each of the mesenchymal cell populations were eliminated by subtracting those transcripts also expressed in at least one of three independent samples of undifferentiated hESCs. Based on this analysis, 1,280 transcripts were selectively expressed in hESMPC-H1.2, 932 transcripts in hESMPC-H9.1, and 1,218 transcripts in primary adult MSCs. A remarkable overlap of 579 transcripts shared among the three mesenchymal populations was observed (D). Using the genes that were selected in the initial filter, we performed a statistical analysis on the expression levels to determine whether the genes were expressed significantly differently in the two cell types. We used a Bayesian extension to the standard t
] to assess this difference. Of the 579 genes, 412 of them were significantly different, at a false discovery rate cutoff of 0.05. The relative fold changes were also extremely large in many of the cases. We also looked at the variance of the expression levels within the cell types. For the MSCs, 94% had a coefficient of variation less than 20% for the expression (log transformed); for the ES-derived cells, 72% had a coefficient of variation less than 20%. Numerous known MSC markers were included in the list of 412 genes, such as the mesenchymal stem cell protein
DSC54 (13.9-fold increase, p
< 0.001), neuropilin 1
(30.4-fold increase, p
< 0.001), hepatocyte growth factor
(48.1-fold increase, p
< 0.001), forkhead box D1
(14.8-fold increase, p
< 0.001), and notch homolog 2
(2.9-fold increase, p
< 0.001) . Table S2
lists the p
-values from the test, the mean and standard deviation of the expression levels, and the relative fold change of all 412 genes between the two types.
Known markers of MSCs, such as mesenchymal stem cell protein DSC54, were all included within the 579 shared transcripts. These findings support the immunocytochemical data and suggest that hESMPCs and primary MSCs are highly related.
MSCs are characterized functionally by their ability to differentiate into mesenchymal tissues, such as fat, cartilage, and bone. Therefore, we tested whether hESMPCs have the same potential ().
Selective Differentiation of hESMPCs into Various Mesenchymal Derivatives
Adipocytic differentiation of hESMPCs was induced under conditions described previously for primary adult MSCs [5
]. Appearance of cells harboring fat granules was observed after 10–14 d in culture. After 3 wk of induction, more than 70% of the cells displayed Oil Red O+ fat granules, and PPARγ,
a marker of adipocytic differentiation, was detected by RT-PCR. (A).
Chondrocytic differentiation was achieved using the pellet culture system [5
]. After 28 d in culture, more than 50% of all cells exhibited robust staining for Alcian Blue, a marker specific for extracellular matrix proteoglycans. Chondrocytic differentiation was confirmed by the gene expression of collagen II
, two components of extracellular matrix selectively expressed by chondrocytes, using RT-PCR (B).
Osteogenic differentiation was induced in the presence of β-glycerolphosphate [5
]. Osteogenesis was demonstrated by specific staining for calcium deposition in the matrix (von Kossa, C; or Alizarin Red, Figure S2
A) and increased expression of bone-specific alkaline phosphatase
and bone sialoprotein
at day 28 of treatment (C and S2
B). At day 28, Alizarin Red staining was detected in approximately 70% of all cells. Throughout these studies, human adult MSCs and foreskin fibroblasts were used as positive and negative controls, respectively.
In addition to adipocytic, chondrocytic, and osteogenic differentiation, reports suggested that adult MSCs can form skeletal muscle [13
]. Although generation of skeletal muscle cells from adult MSCs remains controversial, we tested whether hESMPCs exhibit this potential. Under the conditions previously described [13
], hESMPC-H1.1 and -H9.1 did not yield significant numbers of MyoD+ cells after 15–20 d in culture. However, when confluent cells were maintained in culture in the presence or absence of 5-AzaC without passage for more than 21 d, expression of specific skeletal muscle markers such as MyoD and fast-switch myosin was observed (A). More rapid myogenic differentiation was obtained in the presence of 24-h-conditioned medium from the murine myoblastic cell line C2C12 previously induced to form myotubes [14
]. Direct coculture of hESMPCs with C2C12 cells led to the formation of hESMPC-derived myotubes, as visualized by expression of human nuclear antigen (B), similar to those formed by host C2C12 cells. After 1 wk of coculture, myotubes composed of human nuclei accounted for more than 10% of the total number of human cells present, and each human myotube was composed of up to ten human nuclei. Human cell contribution to myotubes in coculture was confirmed by expression of human muscle-specific transcripts such as MyoD, myosin heavy chain IIa
, and myogenin
(data not shown). These data demonstrate that hESMPCs can give rise to mesenchymal derivatives typically obtained from primary adult MSCs, as well as to cells expressing markers of skeletal muscle.
Myogenic Differentiation of hESMPCs
One concern for the clinical application of hESC-derived progeny in regenerative medicine is the risk of teratoma formation due to the presence of residual undifferentiated ES cells among the differentiated progeny. We did not detect markers of undifferentiated hESCs, such as Nanog
] or Oct-4
], in any of the hESMPCs by RT-PCR (see D) and immunocytochemistry (data not shown), suggesting the lack of any undifferentiated ES cells in hESMPC cultures. However, future in vivo studies are required to rule out the potential of these cells for teratoma formation.