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Interleukin-3-dependent hematopoietic stem cell lines capable of osteoclast formation in vitro.
Recently we reported that the osteoclast originates from the pluripotent hematopoietic stem cell. However, a detailed analysis of the progenitor and precursor stages of the osteoclast lineage is hard to perform with primary cultures of stem cells. In the present investigation interleukin-3 (IL-3)-dependent multipotent hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cells, were assayed for their osteoclast formation capacity. FDCP-mix cell lines A4, C2GM, and 15S were cocultured with periosteum-free 17-day-old fetal metatarsal bones. The effects of culture time, medium composition, and addition of WEHI-3b-conditioned medium (an unpurified IL-3 preparation) on osteoclast formation were studied. 15S cells never differentiated into osteoclasts. Both A4 and C2GM cells were able to generate osteoclasts. Osteoclast formation was visualized by staining for tartrate-resistant acid phosphatase activity and confirmed by 45Ca release assays and electron microscopic studies. Medium supplemented with fetal calf serum clearly supported osteoclast formation from A4 cells better than medium supplemented with cock serum. The difference between fetal calf serum and horse serum is generally less pronounced. C2GM cells formed osteoclasts more readily and, generally, earlier than A4 under all culture conditions. WEHI-3b-conditioned medium addition increased the numbers of osteoclasts and their resorption activity. The coculture of stripped metatarsal bones with FDCP-mix cell lines therefore offers a model system with many possibilities for the study of osteoclastogenesis and its regulation.
Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing.
The field of epidermal stem cells has dramatically advanced in the last decade, leading to a better understanding of the molecular factors, signalling pathways and cellular events that identify and characterize stem cells, thus revealing their immense potential for therapeutic use. Furthermore, multipotent epidermal stem cells present the major advantage of easy accessibility with the discovery of their specific location within the bulge of the hair follicle. This review focuses on the most recent findings on epidermal stem cells, and their potential role in initial epidermal commitment, differentiation and wound healing processes in the skin.
Totipotent migratory stem cells in a hydroid.
Hydroids, members of the most ancient eumetazoan phylum, the Cnidaria, harbor multipotent, migratory stem cells lodged in interstitial spaces of epithelial cells and are therefore referred to as interstitial cells or i-cells. According to traditional understanding, based on studies in Hydra, these i-cells give rise to several cell types such as stinging cells, nerve cells, and germ cells, but not to ectodermal and endodermal epithelial cells; these are considered to constitute separate cell lineages. We show here that, in Hydractinia, the developmental potential of these migratory stem cells is wider than previously anticipated. We eliminated the i-cells from subcloned wild-type animals and subsequently introduced i-cells from mutant clones and vice versa. The mutant donors and the wild-type recipients differed in their sex, growth pattern, and morphology. With time, the recipient underwent a complete conversion into the phenotype and genotype of the donor. Thus, under these experimental conditions the interstitial stem cells of Hydractinia exhibit totipotency.
Forskolin promotes astroglial differentiation of human central neurocytoma cells.
Human central neurocytoma is a kind of the brain tumors that are usually found in anterior part of the lateral ventricles. In this study, we established conditions that allowed proliferation of neurocytoma cells culture and analyzed characteristics of neurocytoma cells in vitro. For in vitro, a condition that used for culturing neural stem cells and contained basic fibroblast growth factor (bFGF) provided high proliferation. RT-PCR analaysis showed that nestin was found in neurocytoma cells, indicating that the neurocytomas possess neural stem cell properties. Interestingly, treatment of neurocytoma cells with forskolin increased expression of glial fibrillary acidic protein with a concomitant decrease in the nestin expression. Forskolin also induced morphological changes of neurocytoma cells to adopt an astrocyte-like phenotype. The results suggest that neurocyotma cells may have properties of multipotent neural stem cells.
Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation.
Bone and bone marrow are closely aligned physiologic compartments, suggesting that these tissues may represent a single functional unit with a common bone marrow progenitor that gives rise to both osteoblasts and hematopoietic cells. Although reports of multilineage engraftment by a single marrow-derived stem cell support this idea, more recent evidence has challenged claims of stem cell transdifferentiation and therefore the existence of a multipotent hematopoietic/osteogenic progenitor cell. Using a repopulation assay in mice, we show here that gene-marked, transplantable marrow cells from the plastic-nonadherent population can generate both functional osteoblasts/osteocytes and hematopoietic cells. Fluorescent in situ hybridization for the X and Y chromosomes and karyotype analysis of cultured osteoblasts confirmed the donor origin of these cells and excluded their generation by a fusion process. Molecular analysis demonstrated a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of seven animals studied, establishing a shared clonal origin for these ostensibly independent cell types. Our findings indicate that the bone marrow contains a primitive cell able to generate both the hematopoietic and osteocytic lineages. Its isolation and characterization may suggest novel treatments for genetic bone diseases and bone injuries.
Behavioral and histological characterization of intrahippocampal grafts of human bone marrow-derived multipotent progenitor cells in neonatal rats with hypoxic-ischemic injury.
Children born with hypoxic-ischemic (HI) brain injury account for a significant number of live births wherein no clinical treatment is available. Limited clinical trials of stem cell therapy have been initiated in a number-of neurological disorders, but the preclinical evidence of a cell-based therapy for neonatal HI injury remains in its infancy. One major postulated mechanism underlying therapeutic benefits of stem cell therapy involves stimulation of endogenous neurogenesis via transplantation of exogenous stem cells. To this end, transplantation has targeted neurogenic sites, such as the hippocampus, for brain protection and repair. The hippocampus has been shown to secrete growth factors, especially during the postnatal period, suggesting that this brain region presents as highly conducive microenvironment for cell survival. Based on its neurogenic and neurotrophic factor-secreting features, the hippocampus stands as an appealing target for stem cell therapy. Here, we investigated the efficacy of intrahippocampal transplantation of multipotent progenitor cells (MPCs), which are pluripotent progenitor cells with the ability to differentiate into a neuronal lineage. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, which involved permanent ligation of the right common carotid artery and subsequent exposure to hypoxic environment. At day 7 after HI injury, animals received stereotaxic hippocampal injections of vehicle or cryopre-served MPCs (thawed just prior to transplantation) derived either from Sprague-Dawley rats (syngeneic) or Fisher rats (allogeneic). All animals were treated with daily immunosuppression throughout the survival period. Behavioral tests were conducted on posttransplantation days 7 and 14 using the elevated body swing test and the rotarod to reveal general and coordinated motor functions. MPC transplanted animals exhibited reduced motor asymmetry and longer time spent on the rotarod than those that received the vehicle infusion. Both syngeneic and allogeneic MPC transplanted injured animals did not significantly differ in their behavioral improvements at both test periods. Immunohistochemical evaluations of graft survival after behavioral testing at day 14 posttransplantation revealed that syngeneic and allogeneic transplanted MPCs survived in the hippocampal region. These results demonstrate for the first time that transplantation of MPCs ameliorated motor deficits associated with HI injury. In view of comparable behavioral recovery produced by syngeneic and allogeneic MPC grafts, allogeneic transplantation poses as a feasible and efficacious cell replacement strategy with direct clinical application. An equally major finding is the observation lending support to the hippocampus as an excellent target brain region for stem cell therapy in treating HI injury.
Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.
Transformed stem cells have been isolated from some human cancers. We report that, unlike other brain cancers, the lethal glioblastoma multiforme contains neural precursors endowed with all of the critical features expected from neural stem cells. Similar, yet not identical, to their normal neural stem cell counterpart, these precursors emerge as unipotent (astroglial) in vivo and multipotent (neuronal-astroglial-oligodendroglial) in culture. More importantly, these cells can act as tumor-founding cells down to the clonal level and can establish tumors that closely resemble the main histologic, cytologic, and architectural features of the human disease, even when challenged through serial transplantation. Thus, cells possessing all of the characteristics expected from tumor neural stem cells seem to be involved in the growth and recurrence of adult human glioblastomas multiforme.
Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation.
Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.
Neuron-to-astrocyte transition: phenotypic fluidity and the formation of hybrid asterons in differentiating neurospheres.
To the extent that their fate choice and differentiation processes can be understood and manipulated, neural stem cells represent a promising therapeutic tool for a variety of neuropathologies. We have previously shown that mature astrocytes possess neural stem cell attributes, and can give rise to neurons through the formation of multipotent neurosphere clones. Here we show that relatively mature neurons generated from neurospheres derived from postnatal subependymal zone or cerebellar cortex undergo a phenotypic transformation into astrocytes that coincides with the appearance of a nonfused, hybrid cell type that shares the morphology, antigenicity, and physiology of both neurons and astrocytes. We refer to this astrocyte/neuron hybrid as an "asteron," and hypothesize that it represents an intermediate step in the trans- or dedifferentiation of neurons into astrocytes. The present finding suggests that seemingly terminally differentiated neural cells may in fact represent points along a bidirectionally fluid continuum of differentiation, with intermediate points represented by "hybrid" cells coexpressing phenotypic markers of more than one lineage.
Does bone marrow contain renal precursor cells?
In the past few years stem cells normally present within the bone marrow have been shown to have considerable plasticity in the repertoire of the cells that they produce. They are capable of producing specific lineages of cells not only of the blood, but also contribute usefully to lineages of other organs. In the kidney, studies in humans and rodents provide evidence for the presence of bone marrow-derived 're-programmed' cells, but it remains possible that the cells that arrive and re-differentiate are no longer stem cells (quiescent cells, not actively proliferating). Nevertheless, the fact that appropriately differentiated cells are delivered deep within the kidney simply by injection of bone marrow cells should make us think differently about the way these organs regenerate and repair. Migratory pathways for multipotential stem cells could be exploited to effect repair using an individual's own stem cells, perhaps after gene therapy. It is clear also that a transplanted organ would in time become affected by the genetic susceptibilities of the recipient, because of phenotypes that are expressed when trafficking cells engraft and differentiate.
The potential of muscle stem cells.
Skeletal muscle contains two types of stem cells: satellite cells, which function as myogenic precursors, and a population of multipotent adult stem cells. Satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. An additional stem cell population in adult muscle displays a remarkable capacity to differentiate into hematopoietic cells as well as muscle following transplantation. This article discusses the characteristics and properties of these cell populations, the relationship between them, and the potential for stem cell-based muscle therapeutics.
Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro.
Neural stem cells from neurogenic regions of mammalian CNS are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem-like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage-specific proteins. The high frequencies of clone-forming cells (about 0.1-10 x 10(-3)) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT-PCR analyses of populations of normal brain- and tumor-derived sister clones revealed transcripts for nestin, neuron-specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor-derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch-signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma-derived clones contain cells immunopositive separately for GFAP or neuronal beta-III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem-like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the developmental pathological heterogeneity of glial tumors.
Neural crest stem cells.
Stem cells are defined by their ability to both self-renew and give rise to multiple lineages in vivo and/or in vitro. As discussed in other chapters in this volume, the embryonic neural crest is a multipotent tissue that gives rise to a plethora of differentiated cell types in the adult organism and is unique to vertebrate embryos. From the point of view of stem cell biology, the neural crest is an ideal source for multipotent adult stem cells. Significant advances have been made in the past few years isolating neural crest stem cell lines that can be maintained in vitro and can give rise to many neural crest derivatives either in vitro or when placed back into the context of an embryo. The initial work identifying these stem cells was carried out with premigratory neural crest from the embryonic neural tube. Later, neural crest stem cells were isolated from postmigratory neural crest, presumably more restricted in developmental potential. More recently it has been demonstrated that neural crest stem cell progenitors persist in the adult in at least two differentiated tissues, the enteric nervous system of the gut and the whisker follicles of the facial skin. In all cases, the properties of the stem cells derived reflect their tissue of origin and the potential of the progenitors becomes more restricted with age. In this chapter we will review this work and speculate on future possibilities with respect to combining our knowledge of neural crest gene function in the embryo and the manipulation of adult neural crest stem cells in vitro and eventually in vivo.
Kinetics of quiescent cord blood stem/progenitor cells with high proliferative potential in stem-cell expansion culture.
BACKGROUND: The most primitive engrafting hematopoietic stem cell (HSC) resides mainly in a tumor growth factor-beta (TGF-beta)-dependent quiescent phase of the cell cycle. In this study, ex vivo expansion of UC blood (UCB) HSCs has been investigated, with the aim of showing whether quiescent HSCs can be recovered from expansion culture. METHODS: AC133(+) stem/progenitor cells from six full term-pregnancies UCB-samples were immunomagnetically selected, followed by ex vivo expansion culture in the presence of thrombopoietin (TPO), c-kit ligand (KL), flt-3 ligand (FL) and IL-6. Quiescent HSCs were detected by a clonogenic assay that allows the detection of multipotent and committed single- lineage quiescent stem/progenitor cells, named mHPP-Q and cHPP-Q, respectively, by means of a TGF-beta blocking Ab. RESULTS: Expansion culture of fresh selected AC133(+) cells for 1 week caused maintenance rather than expansion of mHPP-Q cells and a 1-fold increase in cHPP-Q cells. A further week culture initiated with 7-day expanded AC133(+) cells resulted in an additional 1.5-fold expansion of cHPP-Q while no mHPP-Q cells could be detected. Amplification of cHPP-Q cells in long-term expansion cultures initiated with 14-day expanded AC133(+) cells was observed for at least a further 4 weeks. DISCUSSION: A small proportion of HPP-Q cells recovered from 7-day expansion cultures retain their multilineage potential: longer culturing of these cells results in the loss of multilineage potential while they maintain quiescent behavior and high proliferative potential.
Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction.
Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.
Stem cells, progenitors and myelin repair.
Remyelination, the process by which new myelin sheaths are restored to demyelinated axons, represents one of the most compelling examples of adult multipotent progenitor cells contributing to regeneration of the injured central nervous system (CNS). This process can occur with remarkable efficiency in both clinical disease, such as multiple sclerosis, and in experimental models, revealing an impressive ability of the adult CNS to repair itself. However, the inconsistency of remyelination in multiple sclerosis, and the loss of axonal integrity that results from its failure, makes enhancement of remyelination an important therapeutic objective. Identifying potential targets will depend on a detailed understanding of the cellular and molecular mechanisms of remyelination. In this article we address two important issues. First, we consider the nature of the cell or cells that respond to demyelination and generate new oligodendrocytes, identifying current areas of uncertainty and addressing the role of adult CNS stem and progenitor cells. Second, we discuss the concept of adult progenitor activation following demyelination, focusing on the increased expression of (1) olig transcription factors, (2) bone morphogenetic proteins and (3) fyn, a member of the src-family of tyrosine kinases.
Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development.
All vertebrate embryos have multipotent cells until gastrulation but, to date, derivation of embryonic stem (ES) cell lines has been achieved only for mouse and primates. ES cells are derived from mammalian inner cell mass (ICM) tissue that express the Class V POU domain (PouV) protein Oct4. Loss of Oct4 in mice results in a failure to maintain ICM and consequently an inability to derive ES cells. Here, we show that Oct4 homologues also function in early amphibian development where they act as suppressors of commitment during germ layer specification. Antisense morpholino mediated PouV knockdown in Xenopus embryos resulted in severe posterior truncations and anterior neural defects. Gastrulation stage embryos showed reduced expression of genes associated with uncommitted marginal zone cells, while the expression of markers associated with more mature cell states was expanded. Importantly, we have tested PouV proteins from a number of vertebrate species for the ability to substitute Oct4 in mouse ES cells. PouV domain proteins from both Xenopus and axolotl could support murine ES cell self-renewal but the only identified zebrafish protein in this family could not. Moreover, we found that PouV proteins regulated similar genes in ES cells and Xenopus embryos, and that PouV proteins capable of supporting ES cell self-renewal could also rescue the Xenopus PouV knockdown phenotype. We conclude that the unique ability of Oct4 to maintain ES cell pluripotency is derived from an ancestral function of this class of proteins to maintain multipotency.