Amnion-derived pluripotent/multipotent stem cells

Amniotic epithelium is derived from the epiblast by approx 8 d after fertilization. Other parts of the placenta are derived from extraembryonic tissue. In addition to this developmental difference, amniotic epithelial (AE) cells are known to have unique characteristics, such as low level expression of major histocompatibility complex antigens, and a less restricted differentiation potential. The differentiation of the AE cells to the neural lineage is well documented. Recently, we reported that AE cells from term placenta express several stem cell surface markers that are commonly found on pluripotent stem cells such as embryonic stem cells, and that in culture, AE cells differentiate into cell types from all three germ layers. In this review, we describe the unique characteristics of the AE stem cells and summarize previous work concerning the stem cell nature of cells from amnion.     

Pluripotent and multipotent stem cells in adult tissues

One of the most intriguing questions in stem cell biology is whether pluripotent stem cells exist in adult tissues. Several groups of investigators employing i) various isolation protocols, ii) detection of surface markers, and iii) experimental in vitro and in vivo models, have reported the presence of cells that possess a pluripotent character in adult tissues. Such cells were assigned various operational abbreviations and names in the literature that added confusion to the field and raised the basic question of whether these are truly distinct or overlapping populations of the same primitive stem cells. Unfortunately, these cells were never characterized side-by-side to address this important issue. Nevertheless, taking into consideration their common features described in the literature, it is very likely that various investigators have described overlapping populations of developmentally early stem cells that are closely related. These different populations of stem cells will be reviewed in this paper.     

成体干细胞

在胎儿、儿童和成人组织中存在的多潜能干细胞统称成体干细胞.成体干细胞具有自我更新的能力,并且可以分化成与其来源不同的其他组织类型的细胞.一种组织来源的干细胞可分化成各种类型的细胞,即干细胞可塑性.成体干细胞可塑性是成体干细胞应用于临床的基础,但对干细胞可塑性机制的认识学术界仍存在较大的争议,先后有学者提出脱分化、异质细胞群体和细胞融合等学说,但都不能完全解释成体干细胞的可塑性.近年来的研究提示,成体组织中存在多潜能干细胞,我们发现在胚胎发育后的多种组织中都存在一类原始干细胞群体,在体内、外的特殊环境下,这类原始干细胞可分化为不同胚层的组织细胞,我们称其为亚全能干细胞.亚全能干细胞是存在于人体多种组织中的分化潜能介于从人体胚胎干细胞逐渐形成组织多能干细胞的发育过程中的一种原始干细胞亚群.     

Adult stem cells

Development of a multicellular organism is accomplished through a series of events that are preprogrammed in the genome. These events encompass cellular proliferation, lineage commitment, lineage progression, lineage expression, cellular inhibition, and regulated apoptosis. The sequential progression of cells through these events results in the formation of the differentiated cells, tissues, and organs that constitute an individual. Although most cells progress through this sequence during development, a few cells leave the developmental continuum to become reserve precursor cells. The reserve precursor cells are involved in the continual maintenance and repair of the tissues and organs throughout the life span of the individual. Until recently it was generally assumed that the precursor cells in postnatal individuals were limited to lineage-committed progenitor cells specific for various tissues. However, studies by Young, his colleagues, and others have demonstrated the presence of two categories of precursor cells that reside within the organs and tissues of postnatal animals. These two categories of precursor cells are lineage-committed (multipotent, tripotent, bipotent, and unipotent) progenitor cells and lineage-uncommitted pluripotent (epiblastic-like, ectodermal, mesodermal, and endodermal) stem cells. These reserve precursor cells provide for the continual maintenance and repair of the organism after birth.     

Isolation and characterization of multipotent human keloid-derived mesenchymal-like stem cells

In this study, we report the isolation and characterization of a population of multipotent keloid-derived mesenchymal-like stem cells (KMLSCs) from keloid scalp tissues. These KMLSCs expressed the typical mesenchymal stem cell marker proteins CD13, CD29, CD44, CD90, fibronectin, and vimentin when they were cultured in serum-containing medium and when subsequent exposure to various differentiation media resulted in their differentiation into adipocytes, osteoblasts, chondrocytes, smooth muscle cells, and angiogenic endothelial cells. When KMLSCs were cultured in neural stem culture conditions (i.e., in the presence of epidermal growth factor and fibroblast growth factor 2 in substrate-free conditions), they produced large numbers of neurospheres containing nestin-, CD133-, and SOX2-positive cells that expressed neural-crest stem cell markers. Subsequent exposure of these cells to different differentiation conditions resulted in cells that expressed neuronal cell-, astrocyte-, oligodendrocyte-, or Schwann cell-specific markers. Our study suggests that KMLSCs may be an alternative adult stem cell resource for regenerative tissue repair and auto-transplantation.     

脐带多能干细胞的分离与鉴定

目的 探讨人脐带多能干细胞的分离、扩增方法,鉴定其免疫表型与分化潜能.方法 用Ⅰ型胶原酶消化华通胶(Wharton's jelly)获取细胞,用L-DMEM培养,MTT检测细胞增殖曲线,流式细胞仪检测免疫表型与细胞周期,向脂肪、骨及软骨方向诱导分化,并通过RT-PCR及特异性染色鉴定其分化能力.结果 人脐带华通胶中可获得成纤维样细胞,原代培养后12～16 d达80%～90%融合,传代后可维持未分化状态并稳定增殖,细胞倍增时间为(31.11±2.45)h,体外增殖达10代以上.细胞群中约85.84%处于G0/G1期,S G2 M期占14.16%.表面标记分析显示:CD29、CD44、CD73(SH3)、CDgO、CD105(SH2)阳性,SSEA-4弱阳性,CD31、CD34、CD45和HLA-DR阴性.体外诱导实验表明:细胞具有体外成脂肪、成软骨和成骨能力.结论 脐带是成体干细胞的新来源,从脐带中分离的多能干细胞具有间充质干细胞的生物学特性.     

Unique dielectric properties distinguish stem cells and their differentiated progeny

The relatively new field of stem cell biology is hampered by a lack of sufficient means to accurately determine the phenotype of cells. Cell-type-specific markers, such as cell surface proteins used for flow cytometry or fluorescence-activated cell sorting, are limited and often recognize multiple members of a stem cell lineage. We sought to develop a complementary approach that would be less dependent on the identification of particular markers for the subpopulations of cells and would instead measure their overall character. We tested whether a microfluidic system using dielectrophoresis (DEP), which induces a frequency-dependent dipole in cells, would be useful for characterizing stem cells and their differentiated progeny. We found that populations of mouse neural stem/precursor cells (NSPCs), differentiated neurons, and differentiated astrocytes had different dielectric properties revealed by DEP. By isolating NSPCs from developmental ages at which they are more likely to generate neurons, or astrocytes, we were able to show that a shift in dielectric property reflecting their fate bias precedes detectable marker expression in these cells and identifies specific progenitor populations. In addition, experimental data and mathematical modeling suggest that DEP curve parameters can indicate cell heterogeneity in mixed cultures. These findings provide evidence for a whole cell property that reflects stem cell fate bias and establish DEP as a tool with unique capabilities for interrogating, characterizing, and sorting stem cells.     

Adult stem cells and their cardiac potential

Adult cardiac muscle is unable to repair itself following severe disease or injury. Because of this fundamental property of the myocardium, it was long believed that the adult myocardium is a postmitotic tissue. Yet, recent studies have indicated that new cardiac myocytes are generated throughout the life span of an adult and that extracardiac cells can contribute to the renewal of individual cells within the myocardium. In addition, investigations of the phenotypic capacity of adult stem cells have suggested that their potential is not solely restricted to the differentiated cell phenotypes of the source tissue. These observations have great implications for cardiac biology, as stem cells obtained from the bone marrow and other readily accessible adult tissues may serve as a source of replacement cardiac myocytes. In this review, we describe the evidence for these new findings and discuss their implications in context of the continuing controversy over stem cell plasticity.     

The human embryo, but not its yolk sac, generates lympho-myeloid stem cells: mapping multipotent hematopoietic cell fate in intraembryonic mesoderm

We have traced emerging hematopoietic cells along human early ontogeny by culturing embryonic tissue rudiments in the presence of stromal cells that promote myeloid and B cell differentiation, and by assaying T cell potential in the NOD-SCID mouse thymus. Hematogenous potential was present inside the embryo as early as day 19 of development in the absence of detectable CD34+ hematopoietic cells, and spanned both lymphoid and myeloid lineages from day 24 in the splanchnopleural mesoderm and derived aorta where CD34+ progenitors appear at day 27. By contrast, hematopoietic cells arising in the third week yolk sac, as well as their progeny at later stages, were restricted to myelopoiesis and therefore are unlikely to contribute to definitive hematopoiesis in man.     

bFGF enhances the IGFs-mediated pluripotent and differentiation potentials in multipotent stem cells

It has widely been reported that basic fibroblast growth factor (bFGF) promotes proliferation of human stem cells and contributes to the maintenance of their self-renewal capability through repeated replications. In contrast to embryonic stem cells (ESCs), the effects of growth factors on adult stem cells are poorly understood.

In human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs), bFGF is associated with an increased number of proliferating cells. Furthermore, expression levels of ESC markers were increased after treatment with bFGF. bFGF also increased the expression of FGFR, which in turn increased expression of insulin-like growth factor (IGFs). Since IGFs exert autocrine and paracrine effects on stem cells, bFGF-mediated release of IGFs from hUCB-MSCs might enhance FGFR1 and IGF1R expression in neighboring cells. These receptors could subsequently regulate the effects of bFGF and IGFs in adult stem cells.

These results suggest that positive feedback regulation of bFGF and IGFs leads to proliferation of hUCB-MSCs.     

Global and gene-specific histone modification profiles of mouse multipotent adult germline stem cells

We previously reported the generation of multipotent adult germline stem cells (maGSCs) from spermatogonial stem cells (SSCs) isolated from adult mouse testis. In a later study, we substantiated the pluripotency of maGSCs by demonstrating their close similarity to pluripotent male embryonic stem cells (ESCs) at the epigenetic level of global and gene-specific DNA methylation. Here, we extended the comparative epigenetic analysis of maGSCs and male ESCs by investigating the second main epigenetic modification in mammals, i.e. global and gene-specific modifications of histones (H3K4 trimethylation, H3K9 acetylation, H3K9 trimethylation and H3K27 trimethylation). Using immunofluorescence staining, flow cytometry and western blot analysis, we show that maGSCs are very similar to male ESCs with regard to global levels and nuclear distribution patterns of these modifications. Chromatin immunoprecipitation real-time PCR analysis of these modifications at the gene-specific level further revealed modification patterns of the pluripotency marker genes Oct4, Sox2 and Nanog in maGSCs that are nearly identical to those of male ESCs. These genes were enriched for activating histone modifications including H3K4me3 and H3K9ac and depleted of repressive histone modifications including H3K27me3 and H3K9me3. In addition, Hoxa11, a key regulator of early embryonic development showed the ESC-typical bivalent chromatin conformation with enrichment of both the activating H3K4me3 and the repressive H3K27me3 modification also in maGSCs. Collectively, our results demonstrate that maGSCs also closely resemble ESCs with regard to their chromatin state and further evidence their pluripotent nature.     

Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea

We report the presence of neural crest-derived corneal precursors (COPs) that initiate spheres by clonal expansion from a single cell. COPs expressed the stem cell markers nestin, Notch1, Musashi-1, and ABCG2 and showed the side population cell phenotype. COPs were multipotent with the ability to differentiate into adipocytes, chondrocytes, as well as neural cells, as shown by the expression of beta-III-tubulin, glial fibrillary acidic protein, and neurofilament-M. COP spheres prepared from E/nestin-enhanced green fluorescent protein (EGFP) mice showed induction of EGFP expression that was not originally observed in the cornea, indicating activation of the neural-specific nestin second intronic enhancer in culture. COPs were Sca-1(+), CD34(+), CD45(-), and c-kit(-). Numerous GFP(+) cells were observed in the corneas of mice transplanted with whole bone marrow of transgenic mice ubiquitously expressing GFP; however, no GFP(+) COP spheres were initiated from these mice. On the other hand, COP spheres from transgenic mice encoding P0-Cre/Floxed-EGFP as well as Wnt1-Cre/Floxed-EGFP were GFP(+), indicating the neural crest origin of COPs, which was confirmed by the expression of the embryonic neural crest markers Twist, Snail, Slug, and Sox9. Taken together, these data indicate the existence of neural crest-derived, multipotent stem cells in the adult cornea.     

Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin

In skin, multipotent stem cells generate the keratinocytes of the epidermis, sebaceous gland, and hair follicles. In this paper, we show that Tcf3 and Lef1 control these differentiation lineages. In contrast to Lef1, which requires Wnt signaling and stabilized beta-catenin to express the hair-specific keratin genes and control hair differentiation, Tcf3 can act independently of its beta-catenin interacting domain to suppress features of epidermal terminal differentiation, in which Tcf3 is normally shut off, and promote features of the follicle outer root sheath (ORS) and multipotent stem cells (bulge), the compartments which naturally express Tcf3. These aspects of Tcf3's action are dependent on its DNA binding and Groucho repressor-binding domains. In the absence of its beta-catenin interacting domain, Lef1's behavior (Delta NLef1) seems to be markedly distinct from that of Delta NTcf3. Delta NLef1 does not suppress epidermal differentiation and promote ORS/bulge differentiation, but rather suppresses hair differentiation and gives rise to sebocyte differentiation. Taken together, these findings provide powerful evidence that the status of Tcf3/Lef complexes has a key role in controlling cell fate lineages in multipotent skin stem cells.     

Isolation and in vitro cultivation turns cells from exocrine human pancreas into multipotent stem-cells

Several research groups have reported on the existence and in vitro characterization of multipotent stem-cells from the pancreas. However, the origin of these cells remains largely unexplained. Here, we report that in vitro culturing itself can turn adult cells from human exocrine pancreas into a cell population with typical stem cell characteristics. A simple, yet reliable method enabled us to track cell fates: Combining automated continuous observation using time-lapse microscopy with immunocytochemical analyses, we found that a significant fraction of the pancreatic cells (≈14%) can survive trypsination and displays a drastic change in the protein expression profile. After further cultivation, these cells give rise to a heterogeneous cell population with typical multipotent stem cell characteristics; i.e. they proliferate over long time periods and continuously give rise to specialized cells from at least two germ layers. Although we cannot exclude that a rare pre-existing stem cell-type also contributes to the final in vitro-population, the majority of cells must have been arisen from mature pancreatic cells. Our findings indicate that multipotent cells for regenerative medicine, instead of being laboriously isolated, can be generated in large amounts by in vitro de-differentiation.     

Isolation of novel multipotent neural crest-derived stem cells from adult human inferior turbinate

Adult human neural crest-derived stem cells (NCSCs) are of extraordinary high plasticity and promising candidates for the use in regenerative medicine. Here we describe for the first time a novel neural crest-derived stem cell population within the respiratory epithelium of human adult inferior turbinate. In contrast to superior and middle turbinates, high amounts of source material could be isolated from human inferior turbinates. Using minimally-invasive surgery methods isolation is efficient even in older patients. Within their endogenous niche, inferior turbinate stem cells (ITSCs) expressed high levels of nestin, p75(NTR), and S100. Immunoelectron microscopy using anti-p75 antibodies displayed that ITSCs are of glial origin and closely related to nonmyelinating Schwann cells. Cultivated ITSCs were positive for nestin and S100 and the neural crest markers Slug and SOX10. Whole genome microarray analysis showed pronounced differences to human ES cells in respect to pluripotency markers OCT4, SOX2, LIN28, and NANOG, whereas expression of WDR5, KLF4, and c-MYC was nearly similar. ITSCs were able to differentiate into cells with neuro-ectodermal and mesodermal phenotype. Additionally ITSCs are able to survive and perform neural crest typical chain migration in vivo when transplanted into chicken embryos. However ITSCs do not form teratomas in severe combined immunodeficient mice. Finally, we developed a separation strategy based on magnetic cell sorting of p75(NTR) positive ITSCs that formed larger neurospheres and proliferated faster than p75(NTR) negative ITSCs. Taken together our study describes a novel, readily accessible source of multipotent human NCSCs for potential cell-replacement therapy.