Reprogramming after Chromosome Transfer into Mouse Blastomeres

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities [1], suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4⁺ T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic “cloned” animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.     

The immunogenicity and immunomodulatory function of osteogenic cells differentiated from mesenchymal stem cells

Multipotent mesenchymal stem cells (MSC) are reported to be immunoprivileged as well as immunosuppressive. Hence, they are ideal candidates for allogeneic transplantation to induce regeneration of diseased tissues and organs. However, it is not known whether MSC would retain their immunoprivileged and immunomodulatory properties after differentiating into the local cell types of the transplantation site. This study sought to investigate this question with a novel New Zealand White rabbit osteogenesis model. Results showed that osteogenic cells differentiated from MSC (DOC) in vitro did not express the MHC class II molecule, were incapable of inducing allogeneic lymphocyte proliferation in mixed lymphocyte culture or generating CTL, were inhibitory in ongoing lymphocyte proliferation, and secreted anti-inflammatory cytokines (IL-10 and TGF-beta). There was a significantly higher secretion of IL-10 by DOC than that by MSC, while there was no significant difference between the TGF-beta secretion of MSC and DOC in vitro. However, after IFN-gamma treatment, TGF-beta secretion by DOC significantly decreased despite the increased production by MSC. Four weeks after local DOC implantation, despite MHC class II expression, second-set allogeneic skin rejection showed similar survival to first-set allogeneic skin rejection and DOC appeared to function as osteoblasts. In conclusion, DOC retained their immunoprivileged and immunomodulatory properties in vitro, but the latter was lost following transplantation.     

Therapeutic efficiency of spleen or bone marrow CFU in X-irradiated 89Sr marrow-ablated mice.

Experiments were carried out to compare the therapeutic efficiency (TE: number of CFU required to reduce the mortality from 100 to 50 per cent) of spleen or marrow (BM) stem cells (CFU) grafted into lethally irradiated mice (807 rad) which had been previously treated with 89Sr or splenectomized. It was found that during the reconstitution of the haemopoietic organs, the spleen does not provide more than 10 per cent of the functional cells necessary for survival. Besides, the BM-derived CFU growing in 89Sr marrow-ablated mice remain twice as efficient as the spleen-derived ones. Similarly, spleen-derived CFU transplanted into splenectomized mice are half as efficient as BM-derived ones. It may therefore be assumed that haemopoietic stem cells grafted into a foreign microenvironment retain their original kinetics of growth and differentiation during 7 to 10 days after their transplantation.     

Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair

It is not known whether cells derived from stem cells retain their differentiation and morpho-functional properties after long-term cryopreservation. This information is of importance to evaluate their potential for long-term storage with a view to subsequent use in therapy. Here, we describe the morpho-functional properties of dental pulp stem cells (SBP-DPSCs), and of their differentiated osteoblasts, recovered after long-term cryopreservation. After storage for 2 years, we found that stem cells are still capable of differentiation, and that their differentiated cytotypes proliferate and produce woven bone tissue. In addition, cells still express all their respective surface antigens, confirming cellular integrity. In particular, SBP-DPSCs differentiated into pre-osteoblasts, showing diffuse positivity for ALP, BAP, RUNX-2, and calcein. Recovered osteoblasts expressed bone-specific markers and were easily recognizable ultrastructurally, with no alterations observed at this level. In addition, after in vivo transplantation, woven bone converted into a 3D lamellar bone type. Therefore, dental pulp stem cells and their osteoblast-derived cells can be long-term cryopreserved and may prove to be attractive for clinical applications.     

An overview and synopsis of techniques for directing stem cell differentiation in vitro

The majority of studies on stem cell differentiation have so far been based in vivo, on live animal models. The usefulness of such models is limited, since it is much more technically challenging to conduct molecular studies and genetic manipulation on live animal models compared to in vitro cell culture. Hence, it is imperative that efficient protocols for directing stem cell differentiation into well-defined lineages in vitro are developed. The development of such protocols would also be useful for clinical therapy, since it is likely that the transplantation of differentiated stem cells would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated stem cells. The in vitro differentiation of stem cells, prior to transplantation in vivo, would also avoid spontaneous differentiation into undesired lineages at the transplantation site, as well as reduce the risk of teratoma formation, in the case of embryonic stem cells. Hence, this review critically examines the various strategies that could be employed to direct and control stem cell differentiation in vitro.     

Endothelial progenitor cells for postnatal vasculogenesis

In the past decade, researchers have defined committed stem or progenitor cells from various tissues, including bone marrow, peripheral blood, brain, liver, and reproductive organs, in both adult animals and humans. Whereas most cells in adult organs are composed of differentiated cells, which express a variety of specific phenotypic genes adapted to each organ's environment, quiescent stem or progenitor cells are maintained locally or in the systemic circulation and are activated by environmental stimuli for physiological and pathological tissue regeneration. Recently, endothelial progenitor cells (EPCs) were isolated from peripheral blood CD34, Flk-1, or AC133 antigen-positive cells, which are considered to include a hematopoietic stem cell population, and were shown to be incorporated into foci of neovascularization. This finding, that circulating EPCs may home to sites of neovascularization and differentiate into endothelial cells in situ, is consistent with "vasculogenesis," a critical paradigm for embryonic neovascularization, and suggests that vasculogenesis and angiogenesis may constitute complementary mechanisms for postnatal neovascularization. Previous reports demonstrating therapeutic potential of EPC transplantation in animal models of hindlimb and myocardial ischemia opened the way to the clinical application of cell therapy: the replacement of diseased or degenerating cell populations, tissues, and organs. In this review, we summarize biological features of EPCs and speculate on the utility of EPCs for vascular and general medicine. cell transplantation; ischemia; neovascularization; stem cell     

Engraftment and differentiation of embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: growth of processes into the organ of Corti

Hearing loss in mammals is irreversible because cochlear neurons and hair cells do not regenerate. To determine whether we could replace neurons lost to primary neuronal degeneration, we injected EYFP-expressing embryonic stem cell-derived mouse neural progenitor cells into the cochlear nerve trunk in immunosuppressed animals 1 week after destroying the cochlear nerve (spiral ganglion) cells while leaving hair cells intact by ouabain application to the round window at the base of the cochlea in gerbils. At 3 days post transplantation, small grafts were seen that expressed endogenous EYFP and could be immunolabeled for neuron-specific markers. Twelve days after transplantation, the grafts had neurons that extended processes from the nerve core toward the denervated organ of Corti. By 64-98 days, the grafts had sent out abundant processes that occupied a significant portion of the space formerly occupied by the cochlear nerve. The neurites grew in fasciculating bundles projecting through Rosenthal's canal, the former site of spiral ganglion cells, into the osseous spiral lamina and ultimately into the organ of Corti, where they contacted hair cells. Neuronal counts showed a significant increase in neuronal processes near the sensory epithelium, compared to animals that were denervated without subsequent stem cell transplantation. The regeneration of these neurons shows that neurons differentiated from stem cells have the capacity to grow to a specific target in an animal model of neuronal degeneration.     

Fine structural and histochemical study of lingual taste organs of Rana catesbeiana (Anura: Ranidae) transplanted to liver

Autologous tongues were transplanted onto the liver of the anuran Rana catesbeiana, specimens of which were sacrificed at intervals from 6 hr to 5 months after transplantation. Light and electron microscopy as well as histochemistry disclose that the grafts start to organize into cysts after 14 days. The taste organs occur in all grafts irrespective of the age of the graft. All surviving taste organs exhibit intense adenosine triphosphatase (ATPase) activity and contain fluorescent basal cells of the usual type. Ultrastructurally, the taste organs are composed of three distinct types of cells that lack nerves. The taste and basal cells retain the characteristic dense-core granules in their cytoplasm through the experimental periods. The present results suggested that the taste organs of Rana can survive ectopically in the liver for up to 5 months.     

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold’s mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.     

Hematopoiesis and hematopoietic organs in arthropods

Hemocytes (blood cells) are motile cells that move throughout the extracellular space and that exist in all clades of the animal kingdom. Hemocytes play an important role in shaping the extracellular environment and in the immune response. Developmentally, hemocytes are closely related to the epithelial cells lining the vascular system (endothelia) and the body cavity (mesothelia). In vertebrates and insects, common progenitors, called hemangioblasts, give rise to the endothelia and blood cells. In the adult animal, many differentiated hemocytes seem to retain the ability to proliferate; however, in most cases investigated closely, the bulk of hemocyte proliferation takes place in specialized hematopoietic organs. Hematopoietic organs provide an environment where undifferentiated blood stem cells are able to self-renew, and at the same time generate offspring that differentiate into different blood cell types. Hematopoiesis in vertebrates, taking place in the bone marrow, has been subject to intensive research by immunologists and stem cell biologists. Much less is known about blood cell formation in invertebrate animals. In this review, we will survey structural and functional properties of invertebrate hematopoietic organs, with a main focus on insects and other arthropod taxa. We will then discuss similarities, at the molecular and structural level, that are apparent when comparing the development of blood cells in hematopoietic organs of vertebrates and arthropods. Our comparative review is intended to elucidate aspects of the biology of blood stem cells that are more easily missed when focusing on one or a few model species.     

Understanding cancer stem cell heterogeneity and plasticity

Heterogeneity is an omnipresent feature of mammalian cells in vitro and in vivo. It has been recently realized that even mouse and human embryonic stem cells under the best culture conditions are heterogeneous containing pluripotent as well as partially committed cells. Somatic stem cells in adult organs are also heterogeneous, containing many subpopulations of self-renewing cells with distinct regenerative capacity. The differentiated progeny of adult stem cells also retain significant developmental plasticity that can be induced by a wide variety of experimental approaches. Like normal stem cells, recent data suggest that cancer stem cells (CSCs) similarly display significant phenotypic and functional heterogeneity, and that the CSC progeny can manifest diverse plasticity. Here, I discuss CSC heterogeneity and plasticity in the context of tumor development and progression, and by comparing with normal stem cell development. Appreciation of cancer cell plasticity entails a revision to the earlier concept that only the tumorigenic subset in the tumor needs to be targeted. By understanding the interrelationship between CSCs and their differentiated progeny, we can hope to develop better therapeutic regimens that can prevent the emergence of tumor cell variants that are able to found a new tumor and distant metastases.     

The importance of adipose-derived stem cells and vascularized tissue regeneration in the field of tissue transplantation

The importance of stem cells in regenerating or repairing damaged or diseased tissues is well established, but three factors have to be considered in employing stem cells clinically. The first is how to harvest, handle, and multiply them non-invasively, easily, and effectively. From this standpoint, adipose-derived stem cells are considered to be the best to work with among mesenchymal stem cells; since they were first reported in 2001, their pluripotency, proliferative efficiency, and low donor morbidity have been amply confirmed. The second factor is how to differentiate stem cells into the required cells and use them effectively to construct three-dimensional tissues; here, tissue-specific scaffolds and signaling systems are essential. The third factor is how to ensure survival of the differentiated cells and regenerated tissues. Regenerated tissues need to contain vascular systems to allow both the tissues themselves and the differentiated cells to survive. Thus, we believe that the vascularization of regenerated tissues will be an important field of research in the near future. In this paper, we focus on adipose-derived stem cells and vascularized tissue regeneration within the context of tissue transplantation.     

Engraftment and differentiation of embryonic stem cell–derived neural progenitor cells in the cochlear nerve trunk: Growth of processes into the organ of corti

Hearing loss in mammals is irreversible because cochlear neurons and hair cells do not regenerate. To determine whether we could replace neurons lost to primary neuronal degeneration, we injected EYFP‐expressing embryonic stem cell–derived mouse neural progenitor cells into the cochlear nerve trunk in immunosuppressed animals 1 week after destroying the cochlear nerve (spiral ganglion) cells while leaving hair cells intact by ouabain application to the round window at the base of the cochlea in gerbils. At 3 days post transplantation, small grafts were seen that expressed endogenous EYFP and could be immunolabeled for neuron‐specific markers. Twelve days after transplantation, the grafts had neurons that extended processes from the nerve core toward the denervated organ of Corti. By 64–98 days, the grafts had sent out abundant processes that occupied a significant portion of the space formerly occupied by the cochlear nerve. The neurites grew in fasciculating bundles projecting through Rosenthal's canal, the former site of spiral ganglion cells, into the osseous spiral lamina and ultimately into the organ of Corti, where they contacted hair cells. Neuronal counts showed a significant increase in neuronal processes near the sensory epithelium, compared to animals that were denervated without subsequent stem cell transplantation. The regeneration of these neurons shows that neurons differentiated from stem cells have the capacity to grow to a specific target in an animal model of neuronal degeneration. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

How to make pancreatic beta cells--prospects for cell therapy in diabetes

One promising approach for the cure of diabetes is the replacement of lost insulin-expressing beta cells by cell or regenerative therapy. The recent development of an effective islet transplantation procedure has focused attention on the limiting supply of beta cells. Various sources for new beta cells are therefore being considered, including embryonic stem cells, adult stem cells and transdifferentiation of certain types of differentiated cells, so far with limited success. The major physiological mechanism for adult beta cell formation was recently shown to be beta cell proliferation. This finding underscores the potential use of terminally differentiated beta cells as a starting material for enhancement of beta cell mass.     

干细胞向甲状旁腺细胞的分化及在甲状旁腺功能减退中的研究进展

干细胞是一类能够自我复制、自我更新,具有多向分化潜能,能产生多种分化细胞类型的细胞,由于它的自我复制和多向分化潜能,干细胞技术已经被广泛的应用于细胞移植治疗中。甲状旁腺功能减退作为一种内分泌疾病,目前的治疗方案都不能从根本上治疗该疾病。因此应用干细胞来源的甲状旁腺细胞移植治疗甲状旁腺功能减退越来越受到医学界的重视。目前,已有科研团队报道了应用胚胎干细胞（ESCs）、胸腺上皮细胞和扁桃体间充质细胞向甲状旁腺细胞分化及其在甲状旁腺功能减退中的治疗。本文将干细胞向甲状旁腺细胞的分化及在甲状旁腺功能减退治疗中的研究进展进行综述。     

Fetal Tissue Banking for Transplantation: Characteristics of the Donor Population and Considerations for Donor and Tissue Screening

We initiated this study to evaluate the suitability for therapeutic use in transplantation of tissues obtained from human abortuses. We have developed protocols for the collection, handling and preservation of hepatic stem cells from electively aborted embryos and have developed methods for assessment of the cells so derived and processed. In this paper we present our findings regarding screening of potential donors, acquisition of fetal tissues, and assessment of the tissues for potentially infectious contaminants. We assess the suitability of the tissue donors according to current standards used for donors of commonly transplanted tissues (e.g., bone grafts, skin grafts and heart valves) and present data regarding the real availability of tissues from elective abortion procedures that would meet those standard tissue banking criteria.We specifically evaluated the donor's willingness to provide a blood sample for testing, conducted a detailed interview similar to those used for typical organ and tissue donors, and assessed the type and incidence of contamination in collected tissues. We find that although many women are willing to consent to use of the tissues for transplantation, attrition from the study for various reasons results in few fetal organs ultimately realistically available for transplantation. Typical reasons for attrition include: unwillingness to have a blood sample drawn or tested, positive serology results, social/medical high risk factors for acquisition of transmissible disease, no identifiable organs available, and unacceptable microbial contamination. Thus, although it might seem that due to the numbers of abortions performed annually, that there would be substantial numbers of suitable tissues available, only a small proportion are truly suitable for transplantation.     

Increased Lysis of Stem Cells but Not Their Differentiated Cells by Natural Killer Cells; De-Differentiation or Reprogramming Activates NK Cells

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-γ were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-γ. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells.