Derivation And Characterization Of A Human Embryonic Stem Cell Line And Its Differentiation Into Multipotent Mesenchymal-like Precursors

Human embryonic stem cells (hESCs), derived from inner cell mass (ICM) of preimplantation-stage embryos, possess dual capability to self-renew indefinitely in vitro or give rise to a wide variety of somatic and extraembryonic tissues under certain conditions. Therefore, hESCs attract worldwide attentions serving as an excellent model for in vitro study of human embryonic development, as a unique platform for pharmaceutical screening, and even eventually, as an unlimited resource for cell-based therapies.Currently, considerable attention has focused on the derivation of reliable hESC lines for widespread clinical application. However, the majority of already established hESCs including NIH-approved lines have been directly or indirectly exposed to non-human materials during their derivation and/or propagation, which greatly restrict their future therapeutic potential. Although the human-feeder culture systems, the autogeneic-feeder culture systems and multiple feeder-free culture systems may be safe for cultivation of Undifferentiated hESCs, the ICM isolation procedure free of xeno-products needs to be developed. Here we adopt a non-contact laser-assisted hatching system in combination with sequential culture process to obtain hatched blastocysts as materials for hESC derivation, and derive a hESC line ZJUhES-1 of Chinese population without immunosurgery manipulation that contains non-human materials. The cell line satisfies the criteria of pluripotent hESCs: typically morphological characteristics; the expression of a series of hESC-specific markers; extended proliferative capacity; maintenance of a stable male karyotype after long-term cultivation; robust multiple-lineage differentiation potentials both in vivo and in vitro. Moreover, the cell line has distinct identity revealed from DNA fingerprinting, which is different from previously reported hESC lines.The successful establishment of stem cell-based therapies requires multipotent, immunocompatible stem cells, highly efficient strategies for direct differentiation, and most importantly, well-optimal culture conditions for large-scale expansion of such cell populations. Other than several adult tissues, hESCs represent an alternative infinitely renewable and expansible tissue source for mesenchymal-like precursor isolation. However, poor survival of hESCs after complete dissociation is one major limitation to their current research and prospective clinical application. Here we adopt a selective ROCK inhibitor Y-27632 to promote the survival of completely dissociated hESCs before initiating directed differentiation. Based on the improved monolayer culture system, we reproducibly derive mesenchymal-like precursors from hESCs (hESC-MPs) in chemically defined conditions, without requiring any materials of animal origin. These cells show slim fibroblastic morphology, and satisfy the criteria of MSCs including self-renewal, the expression of multiple MSC-specific markers and the ability to differentiate into mesenchymal lineages such as osteoblasts, adipocytes and chondrocytes. Compared with previously reported hESC-derived MSCs, our hESC-MPs also possess the potentials to differentiate into other representative derivatives of all three embryonic germ layers including smooth muscle cells, cardiomyocytes, functional hepatocytes and neural cells expressing various neurotransmitter phenotypes, making them a better cell source for future clinical application.In addition, we also derive another population of MSCs from hESCs, based on Y-27632-assisted monolayer culture system, in serum-containing conditions. The hESC-MSCs have higher expression of MSC-specific markers and proliferative potential, compared with hESC-MPs derived in chemically defined conditions. When transplanted into mice with carbon tetrachloride (CCl₄)-induced acute liver injury, we observe that these cells can improve liver functions, and stimulate endogenous liver regeneration.