Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.     

Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization

Adults maintain a reservoir of hematopoietic stem cells that can enter the circulation to reach organs in need of regeneration. We developed a novel model of retinal neovascularization in adult mice to examine the role of hematopoietic stem cells in revascularizing ischemic retinas. Adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein. We performed serial long-term transplants, to ensure activity arose from self-renewing stem cells, and single hematopoietic stem-cell transplants to show clonality. After durable hematopoietic engraftment was established, retinal ischemia was induced to promote neovascularization. Our results indicate that self-renewing adult hematopoietic stem cells have functional hemangioblast activity, that is, they can clonally differentiate into all hematopoietic cell lineages as well as endothelial cells that revascularize adult retina. We also show that recruitment of endothelial precursors to sites of ischemic injury has a significant role in neovascularization.     

Totipotent hematopoietic stem cells: normal self-renewal and differentiation after transplantation between mouse fetuses

Successful engraftment of mouse fetal liver cells in early fetal recipients, after microinjection via the placental circulation, is attributable to seeding of the recipient's liver by a cell type that is ancestral to both the myeloid and lymphoid definitive lineages and is capable of sustained self-renewal and differentiation for more than 2 years. This primitive cell is therefore the normal totipotent hematopoietic stem cell (THSC). The use of a large series of mutant anemic recipients with decreasing severity of an endogenous stem-cell defect (W/W, Wv/Wv, Wf/Wf, Wv/+), and therefore of graded selective advantage to normal donor cells, has revealed that engraftment entails marginal numbers of cells--probably individual ones--in the least afflicted hosts. Thus the observed progressive and coordinate shift toward donor-strain erythrocytes, granulocytes and B and T lymphocytes, over time, indicates THSC expansion to form a larger stem-cell pool and normally regulated differentiation of cells from the pool. This transplant system allows allogeneic combinations with impunity and therefore provides many novel experimental possibilities for investigating THSC normal development, genetic abnormalities or neoplastic potential in relation to the intact developmental succession of hematopoietic tissue environments in vivo.     

Endothelial cells mediate the regeneration of hematopoietic stem cells

Recent studies suggest that endothelial cells are a critical component of the normal hematopoietic microenvironment. Therefore, we sought to determine whether primary endothelial cells have the capacity to repair damaged hematopoietic stem cells. Highly purified populations of primary CD31⁺ microvascular endothelial cells isolated from the brain or lung did not express the pan hematopoietic marker CD45, most hematopoietic lineage markers, or the progenitor marker c-kit and did not give rise to hematopoietic cells in vitro or in vivo. Remarkably, the transplantation of small numbers of these microvascular endothelial cells consistently restored hematopoiesis following bone marrow lethal doses of irradiation. Analysis of the peripheral blood of rescued recipients demonstrated that both short-term and long-term multilineage hematopoietic reconstitution was exclusively of host origin. Secondary transplantation studies revealed that microvascular endothelial cell-mediated hematopoietic regeneration also occurs at the level of the hematopoietic stem cell. These findings suggest a potential therapeutic role for microvascular endothelial cells in the self-renewal and repair of adult hematopoietic stem cells.     

Clonal multipotency and effect of long-term in vitro expansion on differentiation potential of human hair follicle derived mesenchymal stem cells

Hair follicle harbors a rich stem cell pool with mesenchymal lineage differentiation potential. Although previous studies with rodent cells demonstrated that hair follicle sheath and papilla cells possess multi-lineage differentiation potential, human hair follicle derived mesenchymal stem cells (hHF-MSCs) have not been characterized in detail in terms of their multipotency. In addition, it is not clear whether these cells are true stem cells that can differentiate along multiple lineages or whether they represent a collection of progenitor cells with restricted differentiation potential. Here we report that hHF-MSCs are highly proliferative cells that can be maintained in culture for ~ 45 population doublings before they start to show signs of cellular senescence. Under appropriate culture conditions, hHF-MSCs differentiated along the myogenic, osteogenic, adipogenic and chondrogenic lineages, as demonstrated by kinetic gene expression profiling and functional assays. Interestingly, the differentiation potential decreased with time in culture in a lineage-specific manner. Specifically, myogenesis and chondrogenesis showed a moderate decrease over time; osteogenesis was maximum at intermediate passages and adipogenesis was highly sensitive to long-term culture and was diminished at late passages. Finally, hHF-MSCs were clonally multipotent as the majority of hHF-MSCs clones (73%) demonstrated bi- or tri-lineage differentiation potential. These results suggest that hHF-MSCs may present as an alternative source of easily accessible, autologous stem cells for tissue engineering and regenerative medicine.     

Functional compensation between hematopoietic stem cell clones in vivo

In most organ systems, regeneration is a coordinated effort that involves many stem cells, but little is known about whether and how individual stem cells compensate for the differentiation deficiencies of other stem cells. Functional compensation is critically important during disease progression and treatment. Here, we show how individual hematopoietic stem cell (HSC) clones heterogeneously compensate for the lymphopoietic deficiencies of other HSCs in a mouse. This compensation rescues the overall blood supply and influences blood cell types outside of the deficient lineages in distinct patterns. We find that highly differentiating HSC clones expand their cell numbers at specific differentiation stages to compensate for the deficiencies of other HSCs. Some of these clones continue to expand after transplantation into secondary recipients. In addition, lymphopoietic compensation involves gene expression changes in HSCs that are characterized by increased lymphoid priming, decreased myeloid priming, and HSC self‐renewal. Our data illustrate how HSC clones coordinate to maintain the overall blood supply. Exploiting the innate compensation capacity of stem cell networks may improve the prognosis and treatment of many diseases.     

Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations

The concept of stem-cell plasticity received strong support from a recent observation that extensively passaged, clonally derived neural stem cells could contribute to hematopoiesis. We investigated whether hematopoietic potential was a consistent or unusual feature of neural stem cells, and whether it depended on the extent of in vitro passaging before transplantation. Here we transplanted over 128 x 10(6) neurosphere cells into 128 host animals; however, we never observed contribution to hematopoiesis, irrespective of the number of passages and despite the use of an assay that could detect the contribution of a single blood stem cell to hematopoietic repopulation. Although extensively cultured neurosphere cells continued to generate neural progeny, marked changes in their growth properties occurred, including changes in growth-factor dependence, cell-cycle kinetics, cell adhesion and gene expression. Our results exclude hematopoietic competence as a consistent property of intravenously infused neural stem cells. However, the consistent changes that occurred during extended passaging are compatible with genetic or epigenetic alterations and suggest that rare transformation events may account for the neural-to-blood fate switch originally reported.     

A stop-EGFP transgenic mouse to detect clonal cell lineages generated by mutation

The investigation of cell lineages and clonal organization in tissues is facilitated by techniques that allow labelling of clonal cell lineages. Here, we describe a novel transgenic mouse that allows clonal cell lineages to be traced in virtually any tissue. A green fluorescent cell lineage is generated by a random mutation at an enhanced green fluorescent protein gene that carries a premature stop codon, ensuring clonality. The transgenic system allows efficient detection of mutations and stem-cell fate mapping in the epidermis using live mice, as well as in the kidney and liver post-mortem. Cell lineages that descended from single epidermal stem cells were found to be capable of generating three adjacent corneocytes using the system, providing evidence for horizontal migration of epidermal cells between epidermal proliferative units (EPUs), in contrast to the classical EPU model. The transgenic mouse system is expected to provide a novel tool for stem-cell lineage studies.     

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

In vitro differentiation of mouse embryonic yolk sac cells

The embryonic yolk sac is the first site in the mammalian embryo in which cells are found that can carry out cell-mediated immune functions, yet the relation of cells of this primitive hematopoietic organ to the development of the mature immune system has not been established. We have initiated a series of experiments to determine the potential of cells of the mouse yolk sac to differentiate in vitro, in order to get an insight into the development of immunocompetence in this primary population of hematopoietic stem cells. The present paper describes the conditions promoting stem-cell differentiation and provides an initial characterization of cell surface phenotypes of the cell lineages established in vitro. Yolk sac cells obtained from 10- to 13-day mouse embryos were maintained in culture for more than 18 months, giving rise to a variety of cell types belonging to the hematopoietic lineages and culminating in the establishment of long-term cell lines. Supernatants of secondary mixed leukocyte cultures were found to be an effective source of growth factors promoting the initial differentiation as well as the maintenance of these cells. Flow-cytometric analysis showed that, in contrast to freshly obtained yolk sac cells, which had no detectable Thy 1 antigen, cells expressing significant levels of Thy 1 were obtained after 1 week or more of culture. Ly1 and Lyt 2 antigens were detected only rarely and the L3T4 (GK 1.5) antigen was never expressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

Estimating the number of hematopoietic or lymphoid stem cells giving rise to clonal chromosome aberrations in blood T lymphocytes

Quantifying the proliferative capacity of long-term hematopoietic stem cells in humans is important for bone marrow transplantation and gene therapy. Obtaining appropriate data is difficult, however, because the experimental tools are limited. We hypothesized that tracking clonal descendants originating from hematopoietic stem cells would be possible if we used clonal chromosome aberrations as unique tags of individual hematopoietic stem cells in vivo. Using FISH, we screened 500 blood T lymphocytes from each of 513 atomic bomb survivors and detected 96 clones composed of at least three cells with identical aberrations. The number of clones was inversely related to their population size, which we interpreted to mean that the progenitor cells were heterogeneous in the number of progeny that they could produce. The absolute number of progenitor cells contributing to the formation of the observed clones was estimated as about two in an unexposed individual. Further, scrutiny of ten clones revealed that lymphocyte clones could originate roughly equally from hematopoietic stem cells or from mature T lymphocytes, thereby suggesting that the estimated two progenitor cells are shared as one hematopoietic stem cell and one mature T cell. Our model predicts that one out of ten people bears a non- aberrant clone comprising >10% of the total lymphocytes, which indicates that clonal expansions are common and probably are not health-threatening.     

Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

Lipocalin 2-mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells

Lipocalin 2 (LCN2), a secreted protein of the lipocalin family, induces apoptosis in some types of cells and inhibits bacterial growth by sequestration of the iron-laden bacterial siderophore. We have recently reported that LCN2 inhibits the production of red blood cells in the mouse. Here we analyzed the role of LCN2 in human hematopoiesis. Expression of LCN2 was observed not only in mature cells such as those of the granulocyte/macrophage and erythroid lineages but also in hematopoietic stem/progenitor cells. We also examined expression of two candidate receptors for LCN2, brain type organic cation transporter (BOCT) and megalin, in various cell types. BOCT showed relatively high levels of expression in erythroid and hematopoietic stem/progenitor cells but lower levels in granulocyte/macrophage and T lymphoid cells. Megalin was expressed at high levels in T lymphoid and erythroid cells but at lower levels in granulocyte/macrophage lineage cells. LCN2 suppressed the growth of erythroid and monocyte/macrophage lineages in vitro, but did not have this effect on cells of other lineages. In addition, immature hematopoietic stem/progenitor cells were not sensitive to LCN2. These results demonstrate a lineage-specific role for LCN2 in human hematopoiesis that is reminiscent of its effects upon mouse hematopoiesis and strongly suggest an important in vivo function of LCN2 in the regulation of human hematopoiesis.     

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.     

The competitive ability of stem cells from mice with Hertwig's anemia

Mice homozygous for the gene, an, have a macrocytic, normochromic anemia. In this report, attempts have been made to cure Hertwig's anemia (an/an) by injecting genetically normal (+/+) stem cells. The anemia of unirradiated an/an mice was alleviated but not completely cured by injection of as many as 3 X 10(7) +/+ bone marrow cells. Lethal irradiation of the an/an recipients was necessary before injections of 10(7) +/+ marrow cells were effective in normalizing the blood parameters. The inability to achieve normal blood values without first destroying the host's own stem cells suggested that the indigenous an/an cells compete effectively with injected +/+ cells. This hypothesis was tested by injecting varying numbers of stem cells from C57BL/6J-+/+ mice, together with stem cells from either WBB6F1-an/an or, as controls, from their WBB6F1-+/+ littermates, into lethally irradiated hosts. The C57BL/6J and WBB6F1 mice have electrophoretically distinguishable hemoglobins. The an/an cells are able to compete in the repopulation of the host hematopoietic tissue as shown by the presence of WBB6F1 hemoglobin in the recipients. The cells from mice with Hertwig's anemia, however, do not compete as effectively as do the same number of cells from the +/+ littermates. These results indicate that the pluripotent hematopoietic stem cells of an/an mice are reduced in number, seeding capacity, or proliferative potential.