Milieu-based versus gene-modulatory strategies for directing stem cell differentiation—A major issue of contention in transplantation medicine

Dear Editor, In recent years, tremendous interest has been aroused over the potential application of both embryonic and adult stem cells in the newly emerging field of regenerative medicine (Hedrick and Daniels, 2003; Gereeht-Nir and hskovitz-Eldor, 2004). However, an essential prerequisite for the application of stem cells in clinical therapy is the development of efficient protocols for committing or directing stem cells into well-defined lineages, before their transplantation in situ (Heng et al., 2004). Undifferentiated or uncommitted stem cells obviously lack expression of specific cell-surface markers that could facilitate their rapid engraftment and integration within the recipient tissue or organ, i.e., gap-junction connexin proteins, surface ligands for interaction with extracellular matrix. Hence, some degree of prior commitment or differentiation of stem ceils into specific lineages likely will enhance the clinical efficacy of transplantation therapy. More importantly, this would reduce greatly the risk of spontaneous differentiation of stem cells into multiple divergent lineages in situ (Mackenzie and Flake, 2001), which could have detrimental effects on the transplant recipient. This is particularly significant in the ease of undifferentiated embryonic stem (ES) cells, which have a demonstrated ability to form teratomas in vivo (Trounson, 2002). It must be noted that there is a significant presence of necrotie/apoptotie ceils, free radicals, and inflammatm'y cytokines within the pathologic environment of damaged or diseased tissues/ organs. This very well could exert an adverse effect on the differentiation pathway of transplanted stem cells that are relatively uncommitted or undifferentiated. For example, in the study of Wang et al. (2001), the transplantation of undifferentiated mesenehymal stem cells into the pathological environment of damaged em'diae muscles resulted in the differentiation of some of these cells into fibroblastie scar tissue, which could in turn impair recovery of heart function after myocardial infarction. Besides clinical therapy, efficient protocols for committing or directing stem cells into welldefined lineages also would be extremely useful for basic scientific research in developmental biology (Eisenberg et al., 2004), as well as tbr pharmacological and cytoxicity screening in vitro (Rohwedel et al., 2001). Two major approaches commit or direct stem cells into a required lineage. First, a milieu-based approach would involve exposing stem cells to a cocktail of exogenous chemicals, cytokines, growth factors, or extraeellular matrix substratum over an extended duration of in vitro culture (Heng et al., 2004). This approach is given further credence by the niche microenvironment theory of stem cell regulation, as proposed by Spradling et al. (200i) who hypothesized that stem cells are strongly dependent on environmental cues for control of cellular fate and lineage determination, rather than evolve complex internal regulatory mechanisms to achieve that purpose. Secondly, a gene-modulatory approach would involve transfection of stem cells, with either recombinant DNA or iRNA, to directly influence gene regulatory networks implicated in cellular fate and lineage determination. First and foremost, a prime consideration is which of these two approaches offers a more efficient means of committing/directing stem cells to a specific lineage. With the milieu-based approach, only a limited fraction of stein ceils is expected to commit and differentiate subsequently into a particular lineage over an extended duration of in vitro culture. This is because the various chemicals, cytokines, growth factors, and ECM substrata within the culture milieu are likely to exert nonspecific pleiotropic effects on stem cell differentiation into multiple lineages. At best, the dosage and combination of various chemicals, cytokines, growth factors, and ECM substrata can be optimized by trial and error to maximize the proportion of stem cells committing and differentiating into a specific lineage while yielding a large number of unrequited lineages. Hence, extensive selection, purification, and proliferation of the cellular lineage of interest would be necessary to achieve the level of purity and cell numbers required for transplantation therapy. This certainly would pose a formidable technical challenge with relatively scarce adult stem cells, which likely have limited proliferative capacity in vitro (Daley et al., 2003). By contrast, a gene-modulatory approach through transfection with either recombinant DNA or iRNA is expected to have a mm-e specific effect on committing/directing stem cell to a required lineage, particularly if there is a resultant upregulation of genes specific to that particular lineage. An added advantage is that genemodulatory strategies could incorporate the use of marker genes to facilitate the selection and purification of specific cell lineages. Commonly used marker genes include green fluorescent protein (GFP) and the neomycin-resistance cassette (Yin et al., 2003; Zandstra et al., 2003). Of particular interest for genetic modulation are transcription factors that exert upstream control of somatic differentiation (Akiyama et al., 2002; Lian et al., 2004; Gerhart et al., 2004). These factors can be thought of as master switches that control the expression of the entire array of proteins that are characteristic or specific to a particular lineage. Indeed, numerous transcription factors have been implicated in early lineage commitment of both adult and embryonic stem cells. Some prominent examples include the role of Sox9 in chondrogenesis (Akiyama et al., 2002), Rtmx2 in osteogenesis (Lian et al., 2004), and MyoD in myogenesis (Gerhart et al., 2004). Howevel, there still is little knowledge on gene-regulatory networks im-