Tejidos

Cytotechnology (2006) 50:181–190 DOI 10.1007/s10616-005-3862-4

Ó Springer 2006

-1

Human embryonic stem cell technology: large scale cell amplification and differentiation
Steve K.W. Oh* and Andre B.H. Choo
Stem Cell Group, Bioprocessing Technology Institute, 20 Biopolis Way, #06 – 01 Centros, Singapore 138668; *Author for correspondence (e-mail: steve_oh@bti.a-star.edu.sg; fax: +65-6478 9561)Received 7 October 2005; accepted 7 October 2005

Key words: Bioreactor, Expansion, Human embryonic stem cell, Pluripotency, Scale-up, Serum-free feeder-free culture

Abstract Embryonic stem cells (ESC) hold the promise of overcoming many diseases as potential sources of, for example, dopaminergic neural cells for Parkinson’s Disease to pancreatic islets to relieve diabetic patients of their dailyinsulin injections. While an embryo has the innate capacity to develop fully functional differentiated tissues; biologists are finding that it is much more complex to derive singular, pure populations of primary cells from the highly versatile ESC from this embryonic parent. Thus, a substantial investment in developing the technologies to expand and differentiate these cells is required in the nextdecade to move this promise into reality. In this review we document the current standard assays for characterising human ESC (hESC), the status of ‘defined’ feeder-free culture conditions for undifferentiated hESC growth, examine the quality controls that will be required to be established for monitoring their growth, review current methods for expansion and differentiation, and speculate on thepossible routes of scaling up the differentiation of hESC to therapeutic quantities. Introduction Embryonic stem cells are pluripotent, capable of differentiation as well as unlimited self-renewal which theoretically make them ideal for replacement, repair or regeneration of tissues and organs. This review covers the current state of the known art and developments in the technologies needed foridentifying and manipulating these versatile cells towards their full potentials. Table 1 (Pera et al. 2000). Typically the characteristic transcription factors include Oct4, Nanog and Sox2; human telomerase and alkaline phosphatase are also highly expressed, along with a panel of cell surface markers including SSEA-3 and SSEA-4, which detect the globoseries glycolipids, and Tra-1-60, Tra-1–81 and GCTM-2which detect proteoglycans such as keratan sulphate. In addition, when human ESC (hESC) are cultured in suspension, they form embryoid body structures which, when left to differentiate without manipulation, can give rise to a variety of phenotypes. These same cells, when injected into severe combined immuno-deficient (SCID) mice, form teratomas which consist of tissues from all three of the germlayers, ecto-, meso- and endoderm. While surface antigen markers have been used for the identification of hESC in various labs, there has

Human embryonic stem cells What is a human embryonic stem cell? There are several important features that define an embryonic stem cell (ESC); these are listed in

182
Table 1. Characteristic markers of hESCs. Transcription factors Surface markers Other markers Invitro differentiation In vivo differentiation (SCID assay) Karyotype Doubling time Oct4, Nanog, Sox2 Stage specific embryonic antigens SSEA-3/4, Tumour related antigens Tra-1-60/1-81, GCTM-2 Human telomerase enzyme, Alkaline phosphatase Neural lineage, Haematopoietic lineage, Cardiac lineage Ectoderm, Mesoderm, Endoderm 23 pairs of chromosomes Typically 30 ± 2 h (Ranges from 24 to 48 h)

not been aconcerted effort to compare a broad range of hESC lines in different labs for the proportion of expression of these markers. It is possible that the different hESC isolates, method of derivation and culture conditions may cause heterogeneity of these markers. Thus the International Stem Cell Initiative led by Prof. Peter Andrews was organized to characterize 77 hESC cell lines from 15 nations with...