Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells

Abstract

Our understanding of how mesodermal tissue is formed has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESCs) is initiated by epithelial-to-mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that the earliest CD326-CD56+ cells, generated from hESCs in the presence of activin A, BMP4, VEGF, and FGF2, represent a multipotent mesoderm-committed progenitor population. CD326-CD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle, and cardiomyocytes, while lacking the pluripotency of hESCs. CD326-CD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a unique approach to study how germ layer specification is regulated and offer a promising target for tissue engineering.

Fig. 1.

Generation of day 3.5 CD326⁻CD56⁺ cells from hESCs occurs during the process of EMT. (A) Flow cytometry analysis of CD326 and CD56 expression in undifferentiated hESCs (H9) at day 0, and cells generated from hESCs after 3.5 d in A-BVF induction conditions. (B) Microarray analysis demonstrates changes in gene expression typical of EMT during the generation of the day 3.5 CD32⁻CD56⁺ population from undifferentiated hESCs. (C) FACS analysis of day 3.5 CD326⁻CD56⁺ cells showing down-regulation of expression of SSEA4 and CD9, two cell surface markers associated with pluripotent phenotype.

Fig. 2.

Time course analysis of expression of the early mesodermal markers KDR, PDGFR-α, and CD34 in the CD326⁻CD56⁺ population. (A) Time course of CD326 and CD56 expression in A-BVF induction conditions (Top). At day 3.5 of culture, CD326⁻CD56⁺ cells were detectable as a distinct population demonstrating lower levels of KDR (Bottom). The control panel shows undifferentiated hESCs that have not been stained with antibody. (B) PDGFR-α and CD56 expression on ungated day 3.5 cells generated in A-BVF (Top). CD34, KDR, and CD56 expression on ungated day 7 cells (Middle row and Lower Left panel). PDGFR-α and KDR expression on CD56⁺ gated cells at day 7 (Lower Right). (C) Subpopulations marked by increased KDR, CD34, and PDGFR-α expression were generated from the CD326⁻CD56⁺ population. Shown are FACS analyses of H9 hESC-derived cultures, representing one of three experiments conducted on different passages of hESCs (using also HES3 and H1 lines). Controls are unstained samples.

Fig. 3.

The CD326⁻CD56⁺ population can generate all mesodermal lineages. CD326⁻CD56⁺ cells were isolated at day 3.5 from hESC cultures in A-BVF conditions and placed in specific differentiation conditions to test mesoderm lineage potential. (A–C) Hematopoietic and endothelial potential as shown by: (A) CD45⁺ and CD235⁺ cells generated after 2 wk of culture on OP9 stroma. (B) Erythroid colony generated in semisolid culture. (C) Generation of CD31⁺CD45⁻ endothelial cells on OP9 stroma. (D) Generation of CD73⁺CD34⁻ mesenchymal cells from day 3.5 CD326⁻CD56⁺ cells after an additional 7-d culture in Matrigel. (E–H) Mesenchymal potential as shown by histochemical staining of (E) cartilage (Alcian blue), (F) bone (von Kossa staining), and (G) adipose (Oil Red O) tissues. (H) Fibroblastic cells generated in day 7 culture. (I) Proportion of GFP⁺ cells, produced after 14 d from day 3.5 GFP⁺CD326⁻CD56⁺ cells, which represent hematopoietic, endothelial, or mesenchymal lineages based on flow cytometry. Data were compiled from limiting dilution analysis as described in Fig. S8. (J and K) Endothelial potential was confirmed, shown on FACS plot (J) by generation of CD31⁺VE-cadherin⁺ endothelial cells after 2 wk in culture. (K) The formation of branching capillaries in Matrigel-coated chamber slides, expressing CD31 (green). (L) Smooth muscle potential shown as cells expressing α-SMA (green). (M–P) Cardiac potential of CD326⁻CD56⁺ cells. (M) Myosin heavy chain, (N) CTNT, and (O) α-actinin protein (all as green) expressing cells at day 14 of culture. TOPRO3-staining (blue) showed cell nuclei. (P) Expression of genes involved in cardiomyocyte specification demonstrated by semiquantitative RT-PCR at day 14 of culture. RPL-7 is a housekeeping gene. Shown are data from H9, HES3, and H1 lines. Magnification 200× in B–H and 400× in K, N, and O).

Fig. 4.

Proposed model of cell surface marker expression during mesodermal specification from hESCs. The initial stage of mesoderm commitment is marked by the process of EMT during which the CD326⁻CD56⁺ population is generated. Subsequent commitment to mesoderm populations with more restricted potential is identified by day 7 of induction cultures by differential expression of the surface markers KDR, PDGFR-α, CD34, and CD73. The phenotype of precursors to the day 7 populations shown is yet to be delineated.