The ACTCellerate Initiative: Large-Scale Combinatorial Cloning of Novel Human Embryonic Stem Cell Derivatives

Abstract

Human embryonic stem cells offer a scalable and renewable source of all somatic cell types. Human embryonic progenitor (hEP) cells are partially differentiated endodermal, mesodermal and ectodermal cell types that have not undergone terminal differentiation and express an embryonic pattern of gene expression. Here, we describe a large-scale and reproducible method of isolating a diverse library of clonally purified hEP cell lines, many of which are capable of extended propagation in vitro. Initial microarray and non-negative matrix factorization gene-expression profiling suggests that the library consists of at least 140 distinct clones and contains many previously uncharacterized cell types derived from all germ layers that display diverse embryo- and site-specific homeobox gene expression. Despite the expression of many oncofetal genes, none of the hEP cell lines tested led to tumor formation when transplanted into immunocompromised mice. All hEP lines studied appear to have a finite replicative lifespan but have longer telomeres than most fetal- or adult-derived cells, thereby facilitating their use in the manufacture of purified lineages for research and human therapy.

A Human Embryonic Stem Cell-Derived Clonal Progenitor Cell Line with Chondrogenic Potential and Markers of Craniofacial Mesenchyme

“…n qRT-PCR-based screen for chondrogenic hEP cell lines in micromass culture Cryopreserved ampules of 100 diverse hESderived embryonic progenitor cell lines [22] at relatively early passages (P8-28) were thawed, cultured, trypsinized and plated under micromass differentiation conditions in the presence of TGF-b3, dexamethasone and insulin, transferrin and selenium, known to induce chondrogenesis in MSCs [7]. After 14 days of differentiation, RNA was prepared from the micromasses, along with bone marrow-derived MSCs differentiated in the same manner (P5-23) and normal human articular chondrocytes.…”

“…#SCR220; Millipore Corp., CA, USA; cat. #ES-84; BioTime, CA, USA) used in this study has been previously described [22]. This line is a derivative of the hES cell line H9 (NIH-registered as WA09).…”

“…The cells lines were maintained at and all subsequent experiments were carried out at 37°C in an atmosphere of 10% CO 2 and 5% O 2 on gelatinized culture vessels, with the relatively high concentration of CO 2 providing physiological pH in relatively low concentrations of O 2 . The hEP cell lines were serially passaged as previously described [22], while confluence was carefully prevented for more than 2 days to prevent differentiation. Cells designated as synchronized in quiescence were plated at a subconfluent density of approximately 25% confluence.…”

“…The clonal hES-derived embryonic progenitor cell lines showed unique gene expression markers in the undifferentiated state, including that of site-specific homeobox genes [22]. The 4D20.8 cell line expressed the relatively rarely expressed perioral homeobox gene LHX8 [24,25].…”

“…In an effort to generate highly purified and sitespecific embryonic progenitors to varied tissues in the human body, and to generate cells capable of differentiating into definitive cartilage lacking markers of hypertrophy, we undertook a combinatorial cloning protocol to create a library of diverse monoclonal hES cell-derived embryonic progenitor cell lines [22]. A preliminary microarray analysis of approximately 200 of these clonal lines showed evidence of more than 140-fold diversity.…”