Catálogo de publicaciones - libros

There are many notions in stem cell biology that lack proof. The stem cell niche is the most typical example. While it is a convenient terminology for designating anything that supports stem cells, the cellular basis of the niche is poorly understood for many stem cell systems. In this chapter, we describe how useful the melanocyte system would be for investigating the molecular and cellular basis of the niche.

We have established several embryonic stem (ES) cell lines of the cynomolgus monkey. They maintain the normal karyotype and pluripotency in culture for long periods. We obtained government approval and grants to produce human ES cell lines from frozen surplus embryos in April 2002. We have established and characterized three human ES cell lines (KhES-1, KhES-2, KhES-3). We started distribution of human ES cells to other research groups in March 2004. It would be important to produce genetically modified monkey and human ES cells for various purposes. After improvement of the transfection and selection methods, we have produced monkey ES cells with integrated transgenes at efficient and reliable rates. We are also investigating reprogramming of somatic cells into pluripotent stem cells by cell fusion with ES cells. Such reprogramming could be used to produce pluripotent stem cells for each patient without therapeutic cloning, which would raise ethical concerns.

Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much optimism for their contribution to human medicine.

Recent studies are beginning to reveal that our basic concepts of epidermal stem cell biology may be based on somewhat tenuous ground. For example, it is often assumed that colony-forming cells represent epidermal stem cells, although this has not proved to be the case in hematopoietic cell lineages. In addition, although most stem cells are not cycling, label-retaining cells are used as a primary measure of epidermal stem cells. Moreover, the locations of stem cell niches in epidermis are still being debated. Finally, while putative stem cell markers abound, the most effective isolation procedure for stem cells has not been determined, and the relative efficiency of various methods of stem cell isolation remains unknown. With a functional assay for epidermal stem cells (analogous to the in-vivo competitive assay used for hematopoiesis), we appear to be in a better position to more clearly define the molecular signature of the true long-term repopulating cell/stem cell of the epidermis. Nonetheless, significant progress has been made in regenerative therapy of the epidermis for ulcer and burn treatment, and for corrective gene therapy for inherited skin diseases

In cases of corneal epithelial stem cell deficiency where ocular surface reconstruction is required, corneal epithelial replacement using a tissue engineering technique shows great potential. Autologous cultivated corneal epithelial stem cell sheets are the safest and most reliable forms of sheet we can use for such treatment; however, they are not useful for treating bilaterally affected ocular surface disorders. In order to treat such cases, we must choose either an allogeneic cultivated corneal epithelial sheet or an autologous cultivated oral mucosal epithelial sheet. If we use the former, the threat of immunological reaction must be dealt with. Therefore, it is imperative that we have a basic understanding of the immunological aspects of ocular surface reconstruction using allogeneic tissues. When using an autologous cultivated oral mucosal epithelial sheet, a basic understanding of ocular surface epithelial biology is required as the sheet is not exactly the same as corneal epithelium.

The Polycomb group (PcG) gene has recently been implicated in the maintenance of hematopoietic stem cells (HSCs). However, the role of each component of PcG complex in HSCs and the impact of forced expression of genes on stem cell self-renewal remain to be elucidated. To address these issues, we performed both loss-of-function and gain-of-function analysis on various PcG proteins. Expression analysis revealed that not only but also other genes are predominantly expressed in HSCs. Loss-of-function analyses, however, demonstrated that absence of is preferentially linked with a profound defect in HSC self-renewal, indicating a central role for Bmi-1, but not the other components, in the maintenance of HSC self-renewal. Overexpression analysis of genes also confirmed an important role of Bmi-1 in HSC self-renewal. Our findings indicate that the expression level of Bmi-1 is the critical determinant for the self-renewal capacity of HSCs. These findings uncover novel aspects of stem cell regulation exerted through epigenetic modifications by the PcG proteins.

We have recently identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells from mouse embryonic stem (ES) cells. Particularly, midbrain THdopaminergic neurons are generated efficiently in this system. These dopaminergic neurons are transplantable and survive well in the 6-OHDA-treated mouse striatum. SDIA induces co-cultured ES cells to differentiate into rostral central nervous system (CNS) tissues containing both ventral and dorsal cells. While early exposure of SDIA-treated ES cells to BMP4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the 4th day of co-culture causes differentiation of neural crest cells and dorsal-most CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic Hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons and HNF3 floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naïve precursors that have the competence of differentiating into the “full” dorsal-ventral range of neuroectodermal derivatives in response to patterning signals. I also discuss the role of SDIA and the mode of rostral-caudal specification of neuralized ES cells. In addition, I would like to discuss them in the light of control of in vitro neural production for the use in regenerative medicine for parkinsonism and retinal degeneration.

The isolation of endothelial progenitor cells (EPCs) derived from bone marrow (BM) was one epoch-making event for the recognition of neovessel formation in adults occurring as physiological and pathological responses. The finding that EPCs home to sites of neovascularization and differentiate into endothelial cells (ECs) in situ is consistent with vasculogenesis, a critical paradigm that has been well described for embryonic neovascularization, but proposed recently in adults in which a reservoir of stem or progenitor cells contribute to vascular organogenesis. EPCs have also been considered as therapeutic agents to supply the potent origin of neovascularization under pathological conditions. This chapter highlights an update of EPC biology as well as its potential use for therapeutic regeneration.