While there is no single way of culturing stem cells or differentiating them into specialized cells, methods are becoming more generalized and widely applicable. For instance, the use of a synthetic serum replacement medium combined with bovine fetal growth factor (bFGF) instead of fetal bovine serum has decreased spontaneous rates of differentiation in a stem cell culture and eliminated many of the lot-to-lot variation problems. With more consistent culture conditions in place, methods for differentiation are becoming more standardized as well. A generalized overview of the current methods for differentiating human embryonic stem cells (hESC) into several important cell types is given below.
Differentiation of Embryonic Stem (ES) and induced pluripotent stem (iPS) cells. ES and iPS cells can form embryoid bodies in cell culture. Embryoid bodies can differentiate into cells of the ectoderm, mesoderm, and endoderm.
The first stage of human embryonic stem cell differentiation is the induction of individual stem cell colonies to form an embryoid body (EB). All downstream differentiated cells are derived from this initial structure. EBs are created by enzymatically detaching undifferentiated stem cell colonies from the dish and transferring them into bFGF-free media (Itskovitz-Eldor et al. 2000). The colonies are kept in suspension and form spherical EBs in about four days. Each EB contains all three germ layers, the ectoderm, mesoderm, and endoderm, just as a developing embryo.
Embryoid bodies after growing in suspension for eight days. Image courtesy of Dr Miguel Esteban.
The central nervous system, hair, and the epidermis are all derived from the ectoderm. The default differentiation pathway for mammalian stem cells is to form neurons. Therefore, the cells of the nervous system have been the most studied of all of the cell types that can be derived from hES cells. There are several protocols for producing neural progenitor cells from undifferentiated cultures. One of these protocols (Zhang et al. 2001) is discussed here.
In order to induce EBs to form neurons, the culture medium is replaced with neural basal media containing bFGF, heparin, and N2 supplement. N2 supplement consists of transferrin, insulin, progesterone, putrescine, and selenite. Two days later, attachment of the differentiating EBs is induced by plating them onto dishes coated with laminin or polyornithine. After an additional 10–11 days in culture, the EBs differentiate into primitive neuroepithelial cells. The identity of the cells can be confirmed by staining for PAX6 (paired box gene 6, a transcription factor), SOX2 (sex determining region Y-box 2, another transcription factor), and N-cadherin (a calcium-dependent cell adhesion molecule specific to neural tissue).
From here it is possible to differentiate the neuroepithelial cells into specific cell types of the central nervous system, including motor neurons (Li et al. 2005), dopaminergic neurons (Yan et al. 2005), and oligodendrocytes (Nistor et al. 2005).
Cells of the mesoderm form most of the body's internal supporting structures, including the blood, muscles, bone, and heart. Because heart disease is a major public health concern, much stem cell research has been devoted to studying cardiomyocytes. Only about 10% of the cells of an EB can form these cells, therefore, the process of producing a pure cardiomyocyte culture involves many rounds of cell isolation. Cardiomyocytes develop spontaneously from 10 day old EBs that have been plated onto gelatin-coated plates (Kehat et al. 2001). Fortunately, cardiomyocytes are easy to identify because of their hallmark rhythmic contractions that can be observed using a phase microscope. Cardiomyocytes can be separated from the rest of the differentiating culture using flow cytometry and antibodies against cardiac markers such as cardiac myosin heavy chain, alpha-actinin, desmin, and cardiac troponin I (Xu et al. 2002).
An alternative method for deriving cardiomyocytes is to transfect a stem cell culture with a viral vector containing a drug-resistance gene driven by the alpha-myosin heavy chain promoter. Subsequent selection for drug resistance enables the selection of cells that are differentiating only into cardiomyocytes (Zhao and Lever 2007).
The differentiation of stem cells into various blood cells has long been of important clinical interest for cancers of the blood, such as leukemia. Much work has been done with hES cells to develop techniques for differentiating them into most of the cells of the hematopoietic system. More information can be found in Keller et al. (1993) and Kaufman et al. (2001).
The endoderm forms many of the internal organs, including the pancreas and the liver. The high rates of diabetes and liver disease have made the production of insulin-secreting cells and hepatocytes key goals in the field of stem cell research.
Diabetes is caused by the destruction of the insulin secreting beta-cells of the Islet of Langerhans in the pancreas. Currently, it is possible to replace these lost cells with those of donors. However, there is very little of this material available for transplantation and the transplantations are not usually a permanent cure, often requiring additional transfusions of beta-cells for the patient to remain asymptomatic. It is now possible to make human embryonic stem cells into all pancreatic cell lineages (Guo and Hebrok 2009). However, the beta-like cells produced during the complex differentiation process are not efficient insulin producers and are not as completely responsive to cell signaling as native beta cells (Furth and Atala 2009). Rapid progress in this line of research combined with the efforts of the induced pluripotent stem cell community make a stem cell–derived treatment for diabetes a possibility in the near future.
In contrast, progress toward developing liver cells (hepatocytes) for transplantation has not been as successful. Stem cells can be differentiated into hepatocyte-like cells using several methods (Hay et al. 2008, Basma et al. 2009, and others). Even though all of these differentiated cells express many of the canonical hepatocyte markers, it is still unclear which of these differentiation techniques and resultant cell types, if any, will be suitable for transplantation (Flohr et al. 2009).
The ability of stem cells to differentiate into nearly any cell in the body gives them the potential to be the source of therapies for many currently incurable illnesses. As research moves forward, standardized techniques for stem cell culture and differentiation will be developed. These new techniques will lay the foundation for future stem cell therapies.
Teratomas composed of tissues derived from the three germ layers produced after injection of the same cell line into immunosuppressed mice. Image courtesy of Dr Miguel Esteban.
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