Manganese-guided cellular MRI of human embryonic stem cell and human bone marrow stromal cell viability.

This study investigated the ability of MnCl(2) as a cellular MRI contrast agent to determine the in vitro viability of human embryonic stem cells (hESC) and human bone marrow stromal cells (hBMSC). Basic MRI parameters including T(1) and T(2) values of MnCl(2)-labeled hESC and hBMSC were measured and viability signal of manganese (Mn(2+))-labeled cells was validated. Furthermore, the biological activity of Ca(2+)-channels was modulated utilizing both Ca(2+)-channel agonist and antagonist to evaluate concomitant signal changes. Metabolic effects of MnCl(2)-labeling were also assessed using assays for cell viability, proliferation, and apoptosis. Finally, in vivo Mn(2+)-guided MRI of the transplanted hESC was successfully achieved and validated by bioluminescence imaging.

Whole genome analysis of human neural stem cells derived from embryonic stem cells and stem and progenitor cells isolated from fetal tissue.

Multipotent neural stem cells (NSC) have been derived from human embryonic stem cells (hESC) as well as isolated from fetal tissues. However, there have been few exclusive markers of NSC identified to date, and the differences between NSC from various sources are poorly understood. Although cells isolated from these two sources share many important characteristics, it is not clear how closely they are related in terms of gene expression. Here, we compare the gene expression profiles of 11 lines of NSC derived from hESC (ES_NSC), four lines of NSC isolated from fetus (F_NSC), and two lines of restricted progenitors in order to characterize these cell populations and identify differences between NSC derived from these two sources. We showed that ES_NSC were clustered together with high transcriptional similarities but were distinguished from F_NSC, oligodendrocyte precursor cells, and astrocyte precursor cells. There were 17 genes expressed in both ES_NSC and F_NSC whose expression was not identified in restricted neural progenitors. Furthermore, the major differences between ES_NSC and F_NSC were mostly observed in genes related to the key neural differentiation pathways. Here, we show that comparison of global gene expression profiles of ES_NSC, F_NSC, and restricted neural progenitor cells makes it possible to identify some of the common characteristics of NSC and differences between similar stem cell populations derived from hESCs or isolated from fetal tissue. Disclosure of potential conflicts of interest is found at the end of this article.

Human embryonic stem cells have a unique epigenetic signature.

Human embryonic stem (hES) cells originate during an embryonic period of active epigenetic remodeling. DNA methylation patterns are likely to be critical for their self-renewal and pluripotence. We compared the DNA methylation status of 1536 CpG sites (from 371 genes) in 14 independently isolated hES cell lines with five other cell types: 24 cancer cell lines, four adult stem cell populations, four lymphoblastoid cell lines, five normal human tissues, and an embryonal carcinoma cell line. We found that the DNA methylation profile clearly distinguished the hES cells from all of the other cell types. A subset of 49 CpG sites from 40 genes contributed most to the differences among cell types. Another set of 25 sites from 23 genes distinguished hES cells from normal differentiated cells and can be used as biomarkers to monitor differentiation. Our results indicate that hES cells have a unique epigenetic signature that may contribute to their developmental potential.

Immunologic consequences of multiple, high-dose administration of allogeneic mesenchymal stem cells to baboons.

Mesenchymal stem cells (MSCs) express low immunogenicity and demonstrate immunomodulatory properties in vitro that may safely allow their transplantation into unrelated immunocompetent recipients without the use of pharmacologic immunosuppression. To test this hypothesis, three groups of baboons (three animals per group) were injected as follows: group 1 animals were injected with vehicle; group 2 animals were injected IV with DiI-labeled MSCs (5 x 106 MSCs/kg body weight) followed 6 weeks later by IM injections of DiO-labeled MSCs (5 x 10(6) MSCs/kg) from the same donor; and group 3 animals were treated similarly as group 2 except that MSCs were derived from two different donors. Muscle biopsies, performed 4 weeks after the second injection of MSCs, showed persistence of DiO-labeled MSCs in 50% of the recipients. Blood was drawn at intervals for evaluation of basic immune parameters (Con A mitogen responsiveness, PBMC phenotyping, immunoglobulin levels), and to determine T-cell and alloantibody responses to donor alloantigens. Host T-cell responses to donor alloantigens were decreased in the majority of recipients without suppressing the overall T-cell response to Con A, or affecting basic parameters of the immune system. All recipient baboons produced alloantibodies that reacted with donor PBMCs. Two of six animals produced alloantibodies that reacted with MSCs. We conclude that multiple administrations of high doses of allogeneic MSCs affected alloreactive immune responses without compromising the overall immune system of recipient baboons. The induction of host T-cell hyporesponsiveness to donor alloantigens may facilitate MSC survival.

Marrow cell transplantation for infantile hypophosphatasia.

An 8-month-old girl who seemed certain to die from the infantile form of hypophosphatasia, an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP), underwent the first trial of bone marrow cell transplantation for this heritable type of rickets. After cytoreduction, she was given T-cell-depleted, haplo-identical marrow from her healthy sister. Chimerism in peripheral blood and bone marrow became 100% donor. Three months later, she was clinically improved, with considerable healing of rickets and generalized skeletal remineralization. However, 6 months post-transplantation, worsening skeletal disease recurred, with partial return of host hematopoiesis. At the age of 21 months, without additional chemotherapy or immunosuppressive treatment, she received a boost of donor marrow cells expanded ex vivo to enrich for stromal cells. Significant, prolonged clinical and radiographic improvement followed soon after. Nevertheless, biochemical features of hypophosphatasia have remained unchanged to date. Skeletal biopsy specimens were not performed. Now, at 6 years of age, she is intelligent and ambulatory but remains small. Among several hypotheses for our patient's survival and progress, the most plausible seems to be the transient and long-term engraftment of sufficient numbers of donor marrow mesenchymal cells, forming functional osteoblasts and perhaps chondrocytes, to ameliorate her skeletal disease.

Ex vivo expansion of bone marrow and cord blood cells to produce stem and progenitor cells for hematopoietic reconstitution.

Ex vivo expansion of a small volume of bone marrow offers an alternate approach to cellular support for repair of a hematopoietic system damaged by radiation exposure. Unpurified bone marrow cells cultured using frequent exchange of medium (perfusion) results in (1) the growth of an adherent layer that provides a supportive microenvironment for hematopoiesis and (2) the growth of hematopoietic stem and progenitor cells. Similar increases in stem and progenitor cells were also observed in cord blood cultures using frequent medium exchange methods. An automated clinical scale system, the AastromReplicell Cell Production System, has been developed to implement the biology of this ex vivo expansion process. Clinical use of ex vivo expanded bone marrow alone or in augmenting a low dose of mobilized peripheral blood stem cells has successfully reconstituted hematopoiesis. Ex vivo expanded cord blood cells combined with unmanipulated cord blood cells have also shown significant benefits in overall survival and engraftment in pediatric and adult patients, respectively. These results suggest that ex vivo expansion of hematopoietic cells provides a mechanism for generating cells capable of hematopoietic reconstitution.