Production of cloned pigs from salivary gland-derived progenitor cells

To achieve tissue stem cell transplantation in clinical settings, translational studies using large animal models are essential to confirm the efficacy and safety of therapy. Therefore, with the ultimate objective of constructing a porcine model of stem cell transplantation in the present study we attempted to clone pigs using porcine salivary gland-derived progenitor cells (pSGPs) as nuclear donors. Normal chromosomal compositions of pSGPs were maintained after five to eight passages (73%, 41 of 56). Cell cycle was efficiently synchronized in G(0)/G(1) phase after 2 days of serum-starved culture (79%). Characteristics of multipotent pSGPs, that is, CD49f and intracellular laminin staining patterns, were unchanged after serum-starved culture. Developmental rate of blastocysts from embryos reconstructed using pSGPs as nuclear donors was significantly higher when compared to embryos reconstructed using fetal fibroblasts (27.7%, 38 of 137 vs. 12.8%, 17 of 138; p < 0.05). When a total of 615 reconstructed embryos were transplanted into four recipient gilts, all gilts became pregnant and produced 12 piglets. These findings suggest that pSGPs represent appropriate donor cells for porcine somatic cell nuclear transfer.     

Transplantation and differentiation of donor cells in the cloned pigs

The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal.     

A novel role for PECAM-1 (CD31) in regulating haematopoietic progenitor cell compartmentalization between the peripheral blood and bone marrow

Although the expression of PECAM-1 (CD31) on vascular and haematopoietic cells within the bone marrow microenvironment has been recognized for some time, its physiological role within this niche remains unexplored. In this study we show that PECAM-1 influences steady state hematopoietic stem cell (HSC) progenitor numbers in the peripheral blood but not the bone marrow compartment. PECAM-1(-/-) mice have higher levels of HSC progenitors in the blood compared to their littermate controls. We show that PECAM-1 is required on both progenitors and bone marrow vascular cells in order for efficient transition between the blood and bone marrow to occur. We have identified key roles for PECAM-1 in both the regulation of HSC migration to the chemokine CXCL12, as well as maintaining levels of the matrix degrading enzyme MMP-9 in the bone marrow vascular niche. Using intravital microscopy and adoptive transfer of either wild type (WT) or PECAM-1(-/-) bone marrow precursors, we demonstrate that the increase in HSC progenitors in the blood is due in part to a reduced ability to migrate from blood to the bone marrow vascular niche. These findings suggest a novel role for PECAM-1 as a regulator of resting homeostatic progenitor cell numbers in the blood.     

The origin of mesoderm in phoronids

Integrin alphaIIb is a cell adhesion molecule expressed in association with beta3 by cells of the megakaryocytic lineage, from committed progenitors to platelets. While it is clear that lymphohemopoietic cells differentiating along other lineages do not express this molecule, it has been questioned whether mammalian hemopoietic stem cells (HSC) and various progenitor cells express it. In this study, we detected alphaIIb expression in midgestation embryo in sites of HSC generation, such as the yolk sac blood islands and the hemopoietic clusters lining the walls of the major arteries, and in sites of HSC migration, such as the fetal liver. Since c-Kit, which plays an essential role in the early stages of hemopoiesis, is expressed by HSC, we studied the expression of the alphaIIb antigen in the c-Kit-positive population from fetal liver and adult bone marrow differentiating in vitro and in vivo into erythromyeloid and lymphocyte lineages. Erythroid and myeloid progenitor activities were found in vitro in the c-Kit(+)alphaIIb(+) cell populations from both origins. On the other hand, a T cell developmental potential has never been considered for c-Kit(+)alphaIIb(+) progenitors, except in the avian model. Using organ cultures of embryonic thymus followed by grafting into athymic nude recipients, we demonstrate herein that populations from murine fetal liver and adult bone marrow contain T lymphocyte progenitors. Migration and maturation of T cells occurred, as shown by the development of both CD4(+)CD8- and CD4-CD8(+) peripheral T cells. Multilineage differentiation, including the B lymphoid lineage, of c-Kit(+)alphaIIb(+) progenitor cells was also shown in vivo in an assay using lethally irradiated congenic recipients. Taken together, these data demonstrate that murine c-Kit(+)alphaIIb(+) progenitor cells have several lineage potentialities since erythroid, myeloid, and lymphoid lineages can be generated.     

Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells.

Bone marrow transplantation to reconstitute defective hematopoietic cell lines in children with congenital defects is limited by donor availability, graft rejection, and graft-versus-host disease (GVHD). These problems can be eliminated by transplanting normal preimmune fetal hematopoietic stem cells (HSC) into an unrelated preimmune fetal recipient. We show here that injections of allogeneic fetal stem cells into preimmune fetal lambs and monkeys result in long-term stable hematopoietic chimerism. HSCs harvested from the livers of preimmune fetal sheep and monkeys when injected into the peritoneal cavity of young unrelated fetal sheep and monkey recipients results in stable, long-term postnatal hematopoietic chimerism involving lymphoid, erythroid, and myeloid cells of donor origin. Donor cell engraftment was achieved without the use of cytoablative procedures and without the development of GVHD.     

The role of the CCN family of proteins in blood cancers

Haematopoiesis is the term used to describe the production of blood cells. This is a tightly regulated hierarchical system in which mature circulating blood cells develop from a small population of haematopoietic stem (HSC) and progenitor cells within the microenvironment of the bone marrow. Molecular and genetic abnormalities arising in these stem cells lead to a block in the normal programme of proliferation and differentiation and result in the development of the blood cancers known as the leukaemias and lymphomas. Recently the regulatory role of the bone marrow microenvironment or niche has also become increasingly recognised. The interface between the bone and bone marrow (endosteum) and the region surrounding the blood vessels (perivascular) provide distinct niches harbouring quiescent HSC or proliferative HSC respectively. Current chemotherapeutic regimes can often successfully target the proliferative HSC but disease relapse occurs due to residual quiescent HSC. Understanding these developmental and regulatory processes and the associated cell communication mechanisms are thus crucial to the development of new treatment strategies. The CCN family of proteins have been recognised to play a key role in all aspects of haematopoiesis.     

Regenerative therapy for neuronal diseases with transplantation of somatic stem cells

Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.     

Heat shock factor 1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of bone marrow stem/progenitor cells

Bone marrow (BM)-derived stem/progenitor cells play an important role in ischemia-induced angiogenesis in cardiovascular diseases. Heat shock factor 1 (HSF1) is known to be induced in response to hypoxia and ischemia. We examined whether HSF1 contributes to ischemia-induced angiogenesis through the mobilization and recruitment of BM-derived stem/progenitor cells using HSF1-knockout (KO) mice. After the induction of ischemia, blood flow and microvessel density in the ischemic hindlimb were significantly lower in the HSF1-KO mice than in the wild-type (WT) mice. The mobilization of BM-derived Sca-1- and c-kit-positive cells in peripheral blood after ischemia was significantly lower in the HSF1-KO mice than in the WT mice. BM stem/progenitor cells from HSF1-KO mice showed a significant decrease in their recruitment to ischemic tissue and in migration, adhesion, and survival when compared with WT mice. Blood flow recovery in the ischemic hindlimb significantly decreased in WT mice receiving BM reconstitution with donor cells from HSF1-KO mice. Conversely, blood flow recovery in the ischemic hindlimb significantly increased in HSF1-KO mice receiving BM reconstitution with donor cells from WT mice. These findings suggest that HSF1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of BM-derived stem/progenitor cells.     

COMPARTMENTS AND CELL FLOWS WITHIN THE MOUSE HAEMOPOIETIC SYSTEM II. ESTIMATED RATES OF INTERCHANGE

Part-body irradiated CBA mice were injected with CBA-T6 bone marrow. In this way a predominantly donor population was established in the femora while the marrow of the humeri remained largely (average 94 %) of host origin. In animals examined cytologically up to 2 years later, no tendency was observed for the proportion of donor cells in the humeri to increase. Splenectomy had no effect on this. When femoral bone marrow from the experimental mice was injected into lethally (whole-body) irradiated recipients, cells originating from the primary host repopulated the lymph nodes to a disproportionate extent. Equilibration between the cell populations of femora and humeri occurred after re-exposure to 600 rad whole-body irradiation, but not after 100 rad or 350 rad; thus, regeneration of damaged bone marrow involved a significant contribution from extrinsic stem cells only after the highest dose of radiation. The data are compatible with an inflow of at most ten effective stem cells per humerus per day from the blood, and suggest a much lower figure. This means that few if any of the stem cells of peripheral blood enter the bone marrow and found haemopoietic clones. Evidence is adduced for the existence of a proliferating lymphoid sub-population in the bone marrow, contributing some 5–10% of the observed mitoses. The mitotic cells in the lymph nodes are replaced from marrow-derived progenitors at an estimated rate of 4–5 %/day. The relevant data for the thymus are more variable, but suggest an average figure of 8–11 %/day. Earlier data from mouse parabionts suggest a lower rate of inflow to the thymus.     

Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs

Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.     

IFN-gamma negatively modulates self-renewal of repopulating human hemopoietic stem cells

Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-gamma, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-gamma might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN-gamma on HSC maintenance and function. Although purified cord blood CD34(+)CD38(-) cells underwent cell divisions in the presence of IFN-gamma, cycling HSCs exposed to IFN-gamma in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN-gamma accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN-gamma can negatively affect human HSC self-renewal.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.     

Isolation of murine and porcine fetal stem cells from somatic tissue

Adult stem cells have been previously isolated from a variety of somatic tissues, including bone marrow and the central nervous system; however, contribution of these cells to the germ line has not been shown. Here we demonstrate that fetal somatic explants contain a subpopulation of somatic stem cells (FSSCs), which can be induced to display features of lineage-uncommitted stem cells. After injection into blastocysts, these cells give rise to a variety of cell types in the resultant chimeric fetuses, including those of the mesodermal lineage; they even migrate into the genital ridge. In vitro, FSSCs exhibit characteristics of embryonic stem cells, including extended self-renewal; expression of stem cell marker genes, such as Pou5f1 (Oct4), Stat3, and Akp2 (Tnap) and growth as multicellular aggregates. We report that fetal tissue contains somatic stem cells with greater potency than previously thought, which might form a new source of stem cells useful in somatic nuclear transfer and cell therapy.     

供体细胞与哺乳动物体细胞核移植

哺乳动物体细胞核移植(克隆)技术在转基因动物生产、珍稀动物资源复原与保护、生物学基础研究等方面业已显示出重要的应用价值,而目前该技术还与诱导多能干细胞技术一同被认为是创制患者特异性多能干细胞,为再生医学临床"细胞治疗"提供素材的最佳手段。但是,体细胞克隆的效率仍不理想,关键机制还不清楚,严重制约了该技术的推广。因此,如何提高克隆效率已成为人们普遍关心的首要问题。在体细胞克隆技术所涉及的各环节中,供体细胞是影响克隆效率的最关键因素之一。该文从供体细胞的生物学因素和技术因素两方面进行了回顾,旨在为进一步探寻建立物种或供体细胞个性化准备方案,为提高动物克隆效率提供参考。     

Reutilization by maternal and embryonal cells of nuclear materials from H3-thymidine labeled lymphocytes introduced into the lumen of intestine of rats

Summary H³-thymidine labeled lymphocytes from thymus and lymph nodes of donor rats were washed and injected in to the intestine of recipient rats on the 11th and 19th day of gestation; subsequent labeling of maternal and embryonal cells was studied autoradiographically 24 hours after injection. In 12-day embryos, numerous stem cells or hemocytoblasts were labeled frequently intensely. In 20-day embryos, stem cells or hemocytoblasts scattered throughout the liver were often labeled. In other fetal tissues at this stage, cells in thymus, spleen, mesenteric lymph node and intestine were labeled but scarcely and weakly. In mothers, labeling in lymphoid tissues was scarce but definite, in thymus, mesenteric lymph node and spleen. These results suggest that nuclear materials from lymphocytes emigrated into the intestinal canal of the mother could be reutilized by maternal and embryonal cells.     

Biology of cord blood cells and future prospects for enhanced clinical benefit

Cord blood (CB) has served as a clinically beneficial source of hematopoietic stem (HSC) and progenitor (HPC) cells for transplantation and correction of a large number of malignant and non-malignant disorders. The capacity of CB to perform these functions is intimately related to the quality and quantity of HSC and HPC present in CB. This review covers the biology of HSC and HPC, efforts to expand these cells ex vivo for enhanced clinical utility that has thus far not been very successful, and recent studies on attempts to enhance the homing and engrafting capability of HSC as an alternative means for more effective use of the limited numbers of CB cells collected. This review also highlights the presence in CB of mesenchymal stem cells, unrestricted somatic stem cells, endothelial progenitor cells and immune cells. The presence and biology of these non-HSC/HPC may open up future possibilities for additional clinical benefit of CB, a product considered mainly for discard before its clinical transplantation potential was realized in the late 1980s.