Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions

Abstract

Chemically defined medium (CDM) conditions for controlling human embryonic stem cell (hESC) fate will not only facilitate the practical application of hESCs in research and therapy but also provide an excellent system for studying the molecular mechanisms underlying self-renewal and differentiation, without the multiple unknown and variable factors associated with feeder cells and serum. Here we report a simple CDM that supports efficient self-renewal of hESCs grown on a Matrigel-coated surface over multiple passages. Expanded hESCs under such conditions maintain expression of multiple hESC-specific markers, retain the characteristic hESC morphology, possess a normal karyotype in vitro, as well as develop teratomas in vivo. Additionally, several growth factors were found to selectively induce monolayer differentiation of hESC cultures toward neural, definitive endoderm͞pancreatic and early cardiac muscle cells, respectively, in our CDM conditions. Therefore, this CDM condition provides a basic platform for further characterization of hESC self-renewal and directed differentiation, as well as the development of novel therapies.chemically defined medium E mbryonic stem cells (ESCs), typically derived from the inner cell mass of the blastocyst, can be propagated indefinitely and differentiated into all of the cell types of the embryo proper (1). ESCs not only hold considerable promise for the treatment of a number of devastating diseases but also provide an excellent system for studying early development and human diseases. Human and mouse ESCs are conventionally maintained in culture with feeder cells and͞or mixtures of exogenous factors (1, 2, 12). However, unknown factors secreted from the feeder cells or contained in the serum may have undesired activities (e.g., inducing differentiation or cell death), which require the addition of other factors that inhibit their effects. Such undefined culture conditions is the main source of inconsistency in large scale and long-term expansion of undifferentiated ESCs. Consequently, the establishment of a well defined culture condition for ESCs would facilitate practical applications of human ESCs (hESCs) and allow for studying and controlling signaling inputs that regulate self-renewal or differentiation of ESCs without multiple unknown and variable factors.Recent studies have shown that feeder-fibroblast conditioned medium (CM) (3), high concentrations of basic FGF (bFGF, 100 ng͞ml) (4) and combinations of bFGF with Noggin (4, 5) or TGF-␤͞activin͞Nodal signaling molecules (6-8) can support longterm culture of hESCs grown on an extracellular matrix (ECM)-coated surface (e.g., Matrigel) in feeder-free conditions. However, these medium conditions typically contain a proprietary serum replacement product, which is a complex mixture of many unknown factors with varying batch-to-batch activities. To come one step closer to a completely defined, animal product-free condition (i.e., chemically defined media and ECM) for long-term self-renewal and efficient clonal expansion of hES...

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

“…However, it is not clear whether the ESCs followed normal developmental sequences in these studies due to a lack of initial specification using developmentally relevant factors known to regulate pancreatic endoderm development. It has recently been shown that treatment of feeder-free hESC colonies with activin directs the differentiation of hESCs into PDX1 + pancreatic endoderm, although the developmental competence of the PDX1 + endoderm was not characterised in this study [8]. In our study, hESC-derived PDX1 + clusters were further differentiated into cells that produce insulin, proinsulin, C-peptide and glucagon upon transplantation.…”

“…To date, only a few studies have suggested the potential of hESCs to differentiate into insulin-producing cells [5][6][7][8]. It was previously reported that insulin-producing cells were found in EBs after spontaneous in vitro differentiation of hESCs [5].…”

“…To date, while many studies have reported that hESCs could be efficiently induced to differentiate into mesodermal and ectodermal lineages, such as cardiomyocytes [1], endothelial cells [2], blood cells [3] and nerve cells [4], few reports have focused on the conversion of hESCs into endodermal lineages, particularly into pancreatic cell types [5][6][7][8], which may provide a limitless stock of insulin-producing cells to treat type 1 diabetes. Moreover, despite increasing knowledge on pancreatic development, there is still debate regarding the origin of beta cells and the markers that can be used to identify their progenitors [9].…”

“…More recently, the generation of pancreatic endodermal cells from hESCs was investigated based on fundamental concepts of early pancreatic specification under spontaneous differentiation [7] and by using chemically defined medium conditions plus activin A [8]. In the present study we investigated whether efficient generation of the pancreatic endoderm can be achieved from hESCs by sequential treatment of developmentally relevant factors using an in vitro system to generate embryoid bodies (EBs).…”

Animal‐cell culture media: History, characteristics, and current issues

“…These cells initially were cultivated on a layer of feeder cells in a serum‐ or KSR‐supplemented medium,104 but in anticipation of clinical applications, the efforts shifted to the development of culture conditions that are feeder‐free and xeno‐free (Table 5). 105, 106, 107, 108, 109, 110, 111 Among these, the E8 medium that was developed by Guokai Chen et al. has become popular.…”