Morphology and cell population kinetics of primary spermatogonia in the frog (Rana esculenta) (Amphibia: Anura)

Two morphologically distinct primary spermatogonial cell types were observed in the frog testis and distinguished on the basis of nuclear characteristics. They have been designated the pale and dark types of primary spermatogonia. On the basis of a kinetic analysis, it is proposed that the pale spermatogonia possess the faculty of self-renewal as well as that of forming dark spermatogonia; they are thus bipotential stem cells comparable to the undifferentiated type of mammalian spermatogonia. The dark spermatogonia, in contrast, are committed to a single pathway, i.e. to form secondary sperrnatogonia, and can be defined as differentiated or committed elements of the primary spermatogonial population. The number of stem cell spermatogonia and differentiated spermatogonia vary according to the period of the year, as does the rate of turnover of stem cells, with nearly 60–90% of cells temporarily out of the cell cycle at any given time. It is indicated that the spermatogonial population represents a 'cell renewal system' in a steady state for appreciably long periods of time, however, changing with season in as far as the magnitude of yield of spermatogonial cells is concerned. This implies that an equality should exist between the rate at which stem cells enter cell-cycling and the rate at which daughter cells change their morphological identity.     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.     

Evidence for a common progenitor of epithelial and mesenchymal components of the liver

Tissues of the adult organism maintain the homeostasis and respond to injury by means of progenitor/stem cell compartments capable to give rise to appropriate progeny. In organs composed by histotypes of different embryological origins (e.g. the liver), the tissue turnover may in theory involve different stem/precursor cells able to respond coordinately to physiological or pathological stimuli. In the liver, a progenitor cell compartment, giving rise to hepatocytes and cholangiocytes, can be activated by chronic injury inhibiting hepatocyte proliferation. The precursor compartment guaranteeing turnover of hepatic stellate cells (HSCs) (perisinusoidal cells implicated with the origin of the liver fibrosis) in adult organ is yet unveiled. We show here that epithelial and mesenchymal liver cells (hepatocytes and HSCs) may arise from a common progenitor. Sca+ murine progenitor cells were found to coexpress markers of epithelial and mesenchymal lineages and to give rise, within few generations, to cells that segregate the lineage-specific markers into two distinct subpopulations. Notably, these progenitor cells, clonally derived, when transplanted in healthy livers, were found to generate epithelial and mesenchymal liver-specific derivatives (i.e. hepatocytes and HSCs) properly integrated in the liver architecture. These evidences suggest the existence of a ‘bona fide'' organ-specific meso-endodermal precursor cell, thus profoundly modifying current models of adult progenitor commitment believed, so far, to be lineage-restricted. Heterotopic transplantations, which confirm the dual differentiation potentiality of those cells, indicates as tissue local cues are necessary to drive a full hepatic differentiation. These data provide first evidences for an adult stem/precursor cell capable to differentiate in both parenchymal and non-parenchymal organ-specific components and candidate the liver as the instructive site for the reservoir compartment of HSC precursors as yet non-localized in the adult.     

ORGANIZATION OF HAEMOPOIETIC STEM CELLS: THE GENERATION-AGE HYPOTHESIS

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 12 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half, to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

Organization of haemopoietic stem cells: the generation-age hypothesis.

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 13 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

Harnessing pluripotency from differentiated cells: a regenerative source for tissue-specific stem cell therapies

Processes involving conversion of mature adult cells into undifferentiated cells have tremendous therapeutic potential in treating a variety of malignant and non-malignant disorders, including degenerative diseases. This can be achieved in autologous or allogeneic settings, by replacing either defective cells or regenerating those that are in deficit through reprogramming more committed cells into stem cells. The concept behind reprogramming differentiated cells to a stem cell state is to enable the switching of development towards the required cell lineage that is capable of correcting the underlying cellular dysfunction. The techniques by which differentiated cells can reverse their development, become pluripotent stem cells and transdifferentiate to give rise to new tissue or an entire organism are currently under intense investigation. Examples of reprogramming differentiation in mature adult cells include nuclear reprogramming of more committed cells using the cytoplasm of empty oocytes obtained from a variety of animal species, or cell surface contact of differentiated cells through receptor ligand interaction. Such ligands include monoclonal antibodies, cytokines or synthetic chemical compounds. Despite controversies surrounding such techniques, the concept behind identification and design/screening of biological or pharmacological compounds to enable re-switching of cell fate in-vivo or ex-vivo is paramount for current drug therapies to be able to target more specifically cellular dysfunction at the tissue/organ level. Herein, this review discusses current research in cellular reprogramming and its potential application in regenerative medicine.     

Maintenance of genetic information in stem cells

The available data on DNA cosegregation in some stem cells are reviewed. Cairns was the first to assume cosegregation of template DNA strands for adult stem cells; i.e., all maternal DNA strands are preserved in one daughter cell, which remains a stem cell, while the newly synthesized DNA strands, which may contain errors, appear in the daughter cell that is committed to differentiation and passes to the transitory compartment of the cell population. The role of asymmetric mitosis in DNA cosegregation and maintenance of genetic information in stem cells is discussed.     

Maintenance of genetic information in stem cells

The available data on DNA cosegregation in some stem cells are reviewed. Cairns was the first to assume cosegregation of template DNA strands for adult stem cells; i.e., all maternal DNA strands are preserved in one daughter cell, which remains a stem cell, while the newly synthesized DNA strands, which may contain errors, appear in the daughter cell that is committed to differentiation and passes to the transitory compartment of the cell population. The role of asymmetric mitosis in DNA cosegregation and maintenance of genetic information in stem cells is discussed.     

Differentiation arrest by hypoxia

The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Although several stromal cells and molecular pathways have been identified, the microenvironment of the stem cell niche remains largely unclear. Recent evidence suggests that stem cells are localized in areas with low oxygen. We have hypothesized that hypoxia maintains the undifferentiated phenotype of stem/precursor cells. In this report, we demonstrate that hypoxia reversibly arrests preadipocytes in an undifferentiated state. Consistent with this observation, hypoxia maintains the expression of pref-1, a key stem/precursor cell gene that negatively regulates adipogenic differentiation. We further demonstrate that the hypoxia-inducible factor-1 (HIF-1) constitutes an important mechanism for the inhibition of adipogenic differentiation by hypoxia. Our findings suggest that hypoxia in the stem cell niche is critical for the maintenance of the undifferentiated stem or precursor cell phenotype.     

Individual Adult Human Neurons Display Aneuploidy: Detection by Fluorescence In Situ Hybridization and Single Neuron PCR

Neurons, once committed, exit the cell cycle and undergo maturation that promote specialized activity and are believed to operate upon a stable genome. We used fluorescence in situ hybridization, selective cell microdissection, and loss of heterozygosity analysis to assess degree of aneuploidy in patients with a neurodegenerative disease and in normal controls. We found that aneuploidy occurs in approximately 40% of mature, adult human neurons in health or disease and may be a physiological mechanism that maintains neuronal fate and function; it does not appear to be an unstable state. The fact that neuronal stem cells can be identified in adult humans and that somatic mosaicism may be found in neuronal precursor cells deserves further investigation before using adult neural stem cells to treat human disease.     

Transdifferentiation, Metaplasia and Tissue Regeneration

Transdifferentiation is defined as the conversion of one cell type to another. It belongs to a wider class of cell type transformations called metaplasias which also includes cases in which stem cells of one tissue type switch to a completely different stem cell. Numerous examples of transdifferentiation exist within the literature. For example, isolated striated muscle of the invertebrate jellyfish (Anthomedusae) has enormous transdifferentiation potential and even functional organs (e.g., tentacles and the feeding organ (manubrium)) can be generated in vitro. In contrast, the potential for transdifferentiation in vertebrates is much reduced, at least under normal (nonpathological) conditions. But despite these limitations, there are some well-documented cases of transdifferentiation occurring in vertebrates. For example, in the newt, the lens of the eye can be formed from the epithelial cells of the iris. Other examples of transdifferentiation include the appearance of hepatic foci in the pancreas, the development of intestinal tissue at the lower end of the oesophagus and the formation of muscle, chondrocytes and neurons from neural precursor cells. Although controversial, recent results also suggest the ability of adult stem cells from different embryological germlayers to produce differentiated cells e.g., mesodermal stem cells forming ecto- or endodermally-derived cell types. This phenomenon may constitute an example of metaplasia. The current review examines in detail some well-documented examples of transdifferentiation, speculates on the potential molecular and cellular mechanisms that underlie the switches in phenotype, together with their significance to organogenesis and regenerative medicine.Key Words: transdifferentiation, metaplasia, tissue regeneration, stem cells, plasticity, reprogramming, regenerative medicine     

Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.     

Intestinal stem cells

The intestinal tract has a rapid epithelial cell turnover, which continues throughout life. The process is regulated and maintained by a population of stem cells, which give rise to all the intestinal epithelial cell lineages. Studies in both the mouse and the human show that these cells are capable of forming clonal crypt populations. Stem cells remain hard to identify, however it is thought that they reside in a 'niche' towards the base of the crypt and their activity is regulated by the paracrine secretion of growth factors and cytokines from surrounding mesenchymal cells. Stem cell division is usually asymmetric with the formation of an identical daughter stem cell and committed progenitor cells. Progenitor cells retain the ability to divide until they terminally differentiate. Occasional symmetric division produces either 2 daughter cells with stem cell loss, or 2 stem cells and eventual clone dominance. This stochastic extinction of stem cell lines with eventual dominance of one cell line is called 'niche succession'. The discovery of plasticity, the ability of stem cells to engraft into, and in some cases replace the function of damaged host tissues has generated a large amount of scientific and clinical interest: however the concept remains controversial and is still a subject of hot debate. Studies are beginning to identify the complex molecular, genetic and cellular pathways underlying stem cell function such as Wnt signalling, bone morphogenetic protein (BMP) and Notch/Delta pathways. The derangement of these pathways within stem cells plays an integral part in the development of malignancy within the intestinal tract.     

Human amniotic epithelial cells maintain mouse spermatogonial stem cells in an undifferentiated state due to high leukemia inhibitor factor (LIF) expression

Spermatogonial stem cells (SSCs), like other stem cells, have unique properties: prolonged proliferation, self-renewal, generation of differentiated progeny, and maintenance of developmental potential. Long-term cultivation of normal SSCs into stable cell lines, and maintaining SSCs in an undifferentiated state capable of self-renewal, is a major challenge. Here, we compare the effect of leukemia inhibitory factor (LIF) expression on mouse SSCs isolated from testicular tissue cultured under different conditions. We found that human amniotic epithelial cells (hAECs) with high LIF expression (LIF(high)) feeder cells allowed mouse SSCs to maintain a high level of AP activity when cultured long term. Expression of some important stem cell markers was higher in mouse SSCs cultured on hAECs (LIF(high)) compared to those cultured on hAECs (LIF(low)). Taken together, these results suggest that LIF expression could be a crucial component for feeder cells to maintain mouse SSCs in an undifferentiated, proliferative state capable of self-renewal.     

Impaired pancreatic development in Hif2-alpha deficient mice

Accumulating data suggest the existence of a link between hypoxia and maintenance of the undifferentiated cell state, but little is known about the cellular signaling mechanisms underlying this process. Recent reports reveal a direct link between components of the hypoxia signaling pathway and Notch pathway in maintaining precursor cells in an undifferentiated state. Here, we report that in the developing mouse pancreas, Hif2-α is expressed in pancreatic progenitor cells, but its expression is lost in committed endocrine progenitors as well as in differentiated endocrine and exocrine cells. In an attempt to analyze the function of HIF2-α in the developing pancreas, we studied Hif2-α^−/− pancreas. Our analyses revealed that in addition to the decreased size and branching, the Hif2-α deficient pancreas also displayed impaired notch signaling and cell differentiation. Finally, we found that HIF2-α binds directly to Notch-IC and that the responsible site for this interaction is within the RAM domain of Notch protein. These results suggest that HIF2-α is required for normal mouse pancreatic development.     

Effects of growth factors on multipotent bone marrow mesenchymal stromal cells

Multipotent bone marrow mesenchymal stromal cells are progenitors of various cell types capable of long-term self-renewal. These cells are an adequate model for studying the most important problems in cell biology, such as self-renewal of stem cells and regulation of their differentiation. Moreover, these cells are a promising resource for regenerative medicine. In this context, isolation of the earliest multipotent mesenchymal stromal cells, their in vitro maintenance in an undifferentiated state, and stimulation of their differentiation in a desired direction appear to be most important. To successfully use the multipotent mesenchymal stromal cells both in fundamental studies and in therapy, it is necessary to modify and standardize the composition of culture medium, replacing blood serum with certain growth factors. These factors have influence on the proliferation and differentiation of most cell types, including multipotent mesenchymal stromal cells. This paper is a review of available data concerning the effects of some growth factors on the multipotent mesenchymal stromal cells of the bone marrow.     

Leukemia stem cell immunophenotyping tool for diagnostic,prognosis, and therapeutics

The existence of cancer stem cells is debatable in numerous solid tumors, yet in leukemia, there is compelling evidence of this cell population. Leukemic stem cells (LSCs) are altered cells in which accumulating genetic and/or epigenetic alterations occur, resulting in the transition between the normal, preleukemic, and leukemic status. These cells do not follow the normal differentiation program; they are arrested in a primitive state but with high proliferation potential, generating undifferentiated blast accumulation and a lack of a mature cell population. The identification of LSCs might guide stem cell biology research and provide key points of distinction between these cells and their normal counterparts. The identification and characterization of the main features of LSCs can be useful as tools for diagnosis and treatment. In this context, the aim of the present review was to connect immunophenotype data in the main types of leukemia to further guide technical improvements.     

The role of stem cells in skeletal and cardiac muscle repair.

In postnatal muscle, skeletal muscle precursors (myoblasts) can be derived from satellite cells (reserve cells located on the surface of mature myofibers) or from cells lying beyond the myofiber, e.g., interstitial connective tissue or bone marrow. Both of these classes of cells may have stem cell properties. In addition, the heretical idea that post-mitotic myonuclei lying within mature myofibers might be able to re-form myoblasts or stem cells is examined and related to recent observations for similar post-mitotic cardiomyocytes. In adult hearts (which previously were not considered capable of repair), the role of replicating endogenous cardiomyocytes and the recruitment of other (stem) cells into cardiomyocytes for new cardiac muscle formation has recently attracted much attention. The relative contribution of these various sources of precursor cells in postnatal muscles and the factors that may enhance stem cell participation in the formation of new skeletal and cardiac muscle in vivo are the focus of this review. We concluded that, although many endogenous cell types can be converted to skeletal muscle, the contribution of non-myogenic cells to the formation of new postnatal skeletal muscle in vivo appears to be negligible. Whether the recruitment of such cells to the myogenic lineage can be significantly enhanced by specific inducers and the appropriate microenvironment is a current topic of intense interest. However, dermal fibroblasts appear promising as a realistic alternative source of exogenous myoblasts for transplantation purposes. For heart muscle, experiments showing the participation of bone marrow-derived stem cells and endothelial cells in the repair of damaged cardiac muscle are encouraging.     

Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous γ-irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after busulphan than after vinblastine even though their proliferation rate is increased. The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan. These results suggest that following cytotoxic agents the agar colony forming cells (“committed” stem cells) are not self-maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.