STEM CELL RESEARCH

Contents

• Introduction
• Identification
• Isolation
• Expansion
• Differentiation
• References

Introduction

Although the "stem cell" concept was introduced decades ago, to date, stem cells can only be defined functionally, not morphologically or phenotypically. Two functions define stem cells. They can be self-renewing and are thus able to propagate and generate additional stem cells. They can also differentiate into various progenitor cells, which commit to further maturation along specific lineages. These functional properties of stem cells have attracted significant interest from both basic and clinical science researchers. The fundamental scheme of stem cells provides a model for basic science researchers to study developmental biology from a very early stage. Stem cell research has also presented opportunities for clinical science in developing new therapies through the functions of repair, replacement and regeneration.

Identification

Since the definition of the stem cell is best given functionally, identification and isolation of this unique cell population have become challenging tasks. Embryonic stem cells (ES), which are derived from the inner cell mass of preimplantation embryos¹ have been recognized as the most pluripotent stem cell population. Successful establishment of human pluripotent ES and embryonic germ (EG) cell lines^2,3 has generated enthusiasm across the field. These cells are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity for differentiation into a wide variety of somatic and extra- embryonic tissues. Although the ES cell has the greatest developmental potential, the ethical dispute revolving around the use of embryonic cells or tissues precludes these cells from experimentation and application. Therefore, adult stem cells may be preferred for future therapeutic applications.

Fig. 1. Stem cells are capable of propagating and generating additional stem cells. They can also differentiate into various progenitor cells, which may then commit to further maturation along specific lineages.

Multiple stem cell populations have been discovered from various adult tissues. One type of adult stem cell population known for its important role in hematopoietic reconstitution is the hematopoietic stem cell (HSC).^4,5 This population may proliferate and differentiate throughout life to produce lymphoid and myeloid cell types. Bone marrow-derived stem cells (BMSC) can differentiate into various cell types: adipocytes, chondrocytes, osteocytes, hepatocytes, cardiomyocytes and neurons.^6-10 The developing and adult mammalian central nervous system contains a population of undifferentiated, multipotent cell precursors, neural stem cells (NSC).¹¹ Discovery of adult neural stem cells¹² and its broad developmental prospective¹³ has also generated vast interest from researchers. The plastic properties of these neural stem cells, identified as nestin positive cells,¹⁴ may be manipulated toward a lineage that synthesizes factors of interest and can be used in grafting strategies to replace substances that are lost after injury or in neurodegenerative diseases. Multilineage cells have also been identified from epithelial and adipose tissues.^15,16 Due to the potential problems of ethical regulation and immuno-incompatibility, autologous adult stem cells may represent a promising option for future tissue engineering strategies. The question of whether adult stem cells can fulfill the same promise as ES cells, however, remains uncertain. Umbilical cord blood (UCB) may be a valid alternative source to bone marrow or peripheral blood for HSC. The characteristics and potential uses of UCB stem cells are currently under extensive examination.

Isolation

Ideally, a true stem cell population needs to be identified in vivo before any in vitro manipulation can occur. Most of the work in isolating stem cells, however, was performed in a relative in vitro, "simulated" format. In vitro technologies based on the recognition of stem cell surface markers, such as CD34,¹⁷ AC133,¹⁸ STRO-1,¹⁹ and neurotrophin receptor p75²⁰ have been used to purify stem cells, including panning, fluorescence-activated cell sorting, immunomagnetic selection and immunoadsorption column separation. Recent evidence casts some uncertainty on the benefits of using a single cell marker in cell isolation for stem cell research until more is known about the novel marker-negative stem cell population. For example, expression of CD34 on the cell membrane does not always correlate with stem cell activity.²¹ In the mouse, there is a large quiescent population of stem cells that lacks CD34 expression yet has full reconstituting capacity. A similar population of dormant CD34-negative hematopoietic stem cells has also been discovered in humans. Therefore, methods designed to achieve removal of specific mature cell lineage markers might prove to be most advantageous in the future.

Expansion

To overcome the limitation of the low frequency of stem cells, researchers have focused on developing protocols for the ex vivo expansion of stem cells. The challenge of maintaining the stem cell's undifferentiated, multilineage potency during its long-term expansion, however, is great. Ideally, an ex vivo stroma- and serum-free system supported with known cytokines and growth factors needs to be developed for clinical applications. Since hematopoietic cells have the potential for providing benefit in a variety of clinical settings, many investigators have explored methods to culture HSC ex vivo to increase the numbers of these cells. Interleukin-3 (IL-3), stem cell factor (SCF), and Flt-3 Ligand (FL) are potential candidates for expansion strategies due to their early acting and lineage-unspecific hematopoietic stimulation. No convincing data has yet been demonstrated, however, on the ex vivo expansion of true HSC.²² The highest expansion of cord blood HSC was obtained with a cocktail containing FL, thrombopoietin (Tpo), IL-6 and IL-11.²³ Platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF) have the greatest ability to support colony growth of human BMSC.²⁴ Neural precursor cells have been successfully isolated from the developing human cortex and expanded in the presence of both EGF and fibroblast growth factor-2 (FGF-2).²⁵ The expansion achieved a 1.5 million-fold increase in precursor cell number over a period of less than 200 days. All of these studies attempting to expand stem cells and progenitor cells in vitro have become possible over the past decade due to the availability of recombinant growth factors and cell selection technologies. Therefore, discoveries of new growth factors or new functions of existing factors could promote the progress of studies in stem cell expansion. Furthermore, a systematic analysis of growth factor receptor expression would be beneficial for designing innovative culture methods for ex vivo expansion of stem cells.

Differentiation

A major focus in stem cell research is the manipulation of cells to differentiate into a targeted population. In the past several years, this field has been flooded with reports on differentiating stem cells into various mature cells both in vitro and in vivo (Figure 1). One of the important extracellular signals that controls stem cell fate is the secretion of growth and differentiation factors. TGF-ß family members have remarkable instructive effects in both ES cell²⁶ and neural crest stem cell differentiation.^27,28 Molecules from the Wnt family also play a significant supportive role in diverse cell differentiation.^29-34 Further research of definitive roles of various Wnt factors is limited, nevertheless, by the lack of accessibility to purified functional Wnt proteins. In addition to secreted factors, integral membrane proteins as well as integrins and the extracelluar matrix also contribute to the microenvironment of stem cells in determining their fate.³⁵

A report of in vitro differentiation of ES cells demonstrated the use of FGF-2, ascorbic acid, sonic hedgehog (SHH) and FGF-8 to obtain dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells.³⁶ The generation of insulin-expressing cells from mouse ES cells, both in vitro and in vivo, has also been reported.³⁷ These studies suggest the feasibility in identifying conditions required for functional mature cell induction of ES cells. Experimental models established from these studies would be useful for understanding the molecular mechanisms behind cellular development and for developing treatments of disorders such as Parkinson's disease and diabetes. Other in vitro and in vivo studies^38,39 have demonstrated the concept that adult stem cells from one tissue can be induced to differentiate into cells of another tissue. The findings imply that there is no intrinsic difference between tissue-specific adult stem cells and ES cells if a novel environment is provided.

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