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PubMed2006

Nonrestrictive developmental regulation of microRNA gene expression.

During different periods of mammalian development, global changes in gene expression occur. Developmental changes in global gene expression have been modeled as a restrictive process. To test the restriction model of global changes in gene expression, we have used embryonic stem (ES) cells as a model system for the early mammalian embryo. ES cells are pluripotent cells that can contribute to all cellular lineages of the developing mammalian fetus and are derived from early embryonic cells. Using this model system, we have studied a new class of RNAs called microRNAs that have been identified and shown to play a role in the direct regulation of messenger RNAs. Here we report the expression signature for 248 microRNAs in 13 independent murine ES cells, embryoid bodies, and somatic tissues. The expression profile for 248 mouse microRNAs was determined for embryonic stem cells, embryoid bodies, mouse embryos, mature heart, lung, liver, kidney, and brain. Characteristic microRNA expression signatures were observed for each evaluated sample. When the characteristic microRNA signatures for developmentally ordered samples were compared, immature samples exhibited a less complex microRNA transcript profile than did mature samples. Our data support a progressive model of microRNA gene expression. Based on the progressive increase in complexity of micro- RNA expression, we hypothesize that the mammalian developmental program requires a temporal coupling of expression between microRNAs and messenger RNAs to enable the developmental potential observed in mammalian ontogeny.

Animals↗
PubMed2006

Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T-cell differentiation culture system.

OBJECTIVE: The OP9-DL1 culture system is an in vitro model for T-cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL-7 has recently emerged. We examined the roles played by IL-7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system. METHODS: Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining. RESULTS: IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T-cell development from the CD4/CD8 double-negative (DN) stage to the CD4/CD8 double-positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single-positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T-lineage differentiation. Flt3 ligand promoted proliferation without affecting differentiation. CONCLUSION: These results validate the OP9-DL1 model for the analysis of T-cell development from bone marrow-derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T-lineage differentiation.

Animals↗
PubMed2006

To make a new intestinal mucosa.

A number of clinical conditions are caused by disorders affecting the mucosal lining of the gastrointestinal tract. Some patients suffer from a loss of mucosal surface area due to congenital defects or due to surgical resections ("short bowel syndrome"). Other patients have inborn or acquired defects of certain mucosal functions (e.g., glucose-galactose malabsorption, bile acid malabsorption). Many patients with these mucosal disorders could be more effectively treated if healthy mucosa were available in larger quantities as a replacement or functional supplement. We therefore developed methods to transplant mucosal stem cells from one part of the intestine to another and to make bioengineered intestinal mucosa. We generated an animal model of bile acid malabsorption using rats that underwent resection of the distal 25% of their small intestine (ileum). This resulted in significant losses of bile acids with the fecal excretions in these animals. We subsequently harvested ileal stem cell clusters from neonatal donors, removed the mucosa from a segment of proximal intestine (jejunum), and implanted the stem cell clusters into the debrided segment of jejunum. After four weeks, the animals had developed a functional "neomucosa." We inserted the "neo-ileal" segment into continuity as a substitute ileum. Postoperative measurements of fecal bile acid excretion showed that we were able to reverse the malabsorption syndrome in this model. This was the first reported neo-mucosa-based treatment of a malabsorption syndrome in vivo. We subsequently studied different biodegradable PGA and PLLA scaffoldings to generate bioengineered intestinal mucosa. We implanted these materials into omentum of rats and were able to identify a PGA/PLLA hybrid material on which engraftment rates of 36% of the available surface area could be achieved. Most recently, we developed a novel technique that permits direct observation of cell-biomaterial interactions after implantation into omentum or intestine in vivo. This method will help to optimize engraftment conditions for stem cell clusters on biomaterials.

Animals↗
PubMed2005

Effect of paclitaxel and mesenchymal stem cells seeding on ex vivo vascular endothelial repair and smooth muscle cells growth.

Late thrombosis and neointima proliferation after paclitaxel-eluting stents implanting may be related to delayed endothelial cells (ECs) regeneration. This study was to investigate whether mesenchymal stem cells (MSCs) seeding can accelerate endothelial repair and attenuate late smooth muscle cells (SMCs) proliferation after paclitaxel intervention. An ex vivo model of endothelium repair was developed in which rabbit smooth muscle cells were inoculated in the upper chamber and rabbit endothelial cells/human mesenchymal stem cells in the lower chamber of a co-culture system. Paclitaxel (10 nmol/L, 20 min) inhibited smooth muscle cell growth of the confluent endothelial cell group during the observed period. However, increased smooth muscle cells growth was observed in the proliferative endothelial cells group 10 days after paclitaxel intervention. Mesenchymal stem cell seeding inhibited late smooth muscle cell growth incompatible with the effect of proliferative endothelial cells. However, no inhibition on smooth muscle cell growth was observed with mesenchymal stem cell seeding in comparison to the effect of confluent endothelial cells. No vWF but Flk-1 protein was observed in the 25.71% of mesenchymal stem cells after having been co-cultured with rabbit endothelial cells for 5 days. These results indicate that late smooth muscle cell proliferation is closely related to the delayed endothelial cells regeneration after paclitaxel application. Mesenchymal stem cell seeding partly attenuates the late smooth muscle cell proliferation. Mesenchymal stem cells co-cultured with mature endothelial cells have the ability to differentiate toward endothelial cells.

Animals↗
PubMed1998

Thiotepa improves allogeneic bone marrow engraftment without enhancing stem cell depletion in irradiated mice.

Thiotepa (TT) has long been considered for inclusion in clinical bone marrow transplant (BMT) conditioning regimens in an attempt to prevent allograft rejection and leukemia relapse. These studies have been encouraged by initial murine experiments showing a clear improvement in allogeneic bone marrow engraftment with addition of TT to total body irradiation (TBI) where it was assumed that TT enhances donor-type chimerism via ablation of competing stem cells in the recipient. The aim of the present study was to re-evaluate the hematological toxicity of TT among different stem cell subsets that included primitive cells capable of long-term repopulation and to assess how the combination of TT with TBI influences the development of donor engraftment in both syngeneic (B6-Gpi-1a --> B6-Gpi-1b) and H-2 compatible allogeneic (BALB.B10 --> B6) BMT models. At 24 h after TT (20 mg/kg) the femoral content of different stem cell subsets was determined from the frequency of transient repopulating, and the more primitive cobblestone area-forming, cells (CAFCs) growing in stroma-supported cultures. This assay showed a large TT-induced depletion (2% survival) of early clones developing at day 7 in culture but survival recovered towards normal for later appearing clones developing from more primitive CAFC subsets. The sparing of these primitive stem cells was reflected as undetectable levels of donor marrow repopulation in recipients given TT followed by syngeneic BMT. Addition of TT to TBI did not significantly improve long-term engraftment of syngeneic marrow while this combination had a dramatic effect in allogeneic BMT by preventing allograft rejection. In this respect TT shares similar properties with cyclophosphamide and suggests that the large improvement of allogeneic stem cell engraftment is attributable to the immune suppressive properties of TT rather than to its toxicity against host primitive stem cells.

Alkylating Agents↗
PubMed1976

Tumor virus effects on immunocyte precursor cells. Hemopoietic stem cell behavior and leukemogenic susceptibility.

Studies of Rauscher virus-induced erythroleukemia have demonstrated immunodepressive effects in the host and enhanced leukemogenesis with adjuvant administration. These observations led to the study of leukemic development in the NZB strain as a natural model of the experimentally adjuvant-stimulated animal. The results of such investigation would attribute the increased susceptibility of NZB mice to the possession of an enlarged population of pluripotent hemopoietic stem cells in active cell cycle. Studies with radiation chimeras have further shown that elevated endogenous spleen colony formation, the increased potential for autoimmunity, and for susceptibility to Rauscher viral leukemogenesis are all linked through the NZB hemopoietic system. It is concluded that the presence of an enlarged compartment of cyclically active stem cells may be an etiologic factor in the susceptibility to both virus-induced leukemia and the development of autoimmune disease.

Adjuvants, Immunologic↗
PubMed2005

Self-correction of chromosomally abnormal embryos in culture and implications for stem cell production.

OBJECTIVE: To ascertain whether embryos classified by preimplantation genetic diagnosis (PGD) for infertility as abnormal and then plated to obtain stem cells would self-correct partially or totally in culture, producing disomic stem cells. DESIGN: Prospective study to determine the chromosome status of embryos on day 3 and 6, as well as cultured cells derived from inner cell masses from the same embryos when cultured up to day 12. SETTING: Research laboratory. PATIENT(S): Patients undergoing PGD of aneuploidy. INTERVENTION(S): Of 142 embryos classified by PGD for aneuploidy as abnormal, 50 were cultured to the blastocyst stage. At that stage a fraction of the embryos underwent trophectoderm biopsy to reconfirm the PGD diagnosis. After further co-culture with feeders up to day 12, 34 embryos attached to the feeder cells. Of those, 24 were analyzed by fluorescence in situ hybridization (FISH) and the rest for the expression of Oct-4, SSEA-3, SSEA-4, TRA1-60, and TRA1-80. MAIN OUTCOME MEASURE(S): Disomic cells obtained from trisomic embryos. RESULT(S): Analysis by FISH of day-12 cultures showed that 7 were totally normal, 6 were mostly abnormal, and 11 had experienced some chromosome normalization, having between 21% and 88% normal cells. Day-12 culture was positive for Oct-4 expression by reverse transcriptase polymerase chain reaction analysis and for SSEA-3, SSEA-4, TRA1-60, and TRA1-80 by immunocytochemistry. CONCLUSION(S): Chromosome self-normalization occurs in a significant proportion of chromosomally abnormal embryos, possibly because of the loss of a chromosome in trisomic cells after blastocyst stage. Thus chromosomally abnormal embryos are a potential source of disomic stem cells. Not all chromosomally abnormal embryos self-corrected. Abnormal stem cells that might be derived could be used as models to study the effect of chromosomal abnormalities on human development.

Chromosome Aberrations↗
PubMed2001

cAMP is involved in the differentiation of human teratocarcinoma cells.

The generation of fully differentiated post-mitotic human neuronal cells from stem cells (human teratocarcinoma (hNT2) cells) might enable the development of a co-culture model of human neurons with human Schwann cells (SCs). This co-culture model is an important tool to study formation of myelin sheaths. However, the thin process of the post-mitotic human neuronal cells formed under known culture conditions do not provide a good substrate for human SCs to start myelination. We optimized the culture conditions of these cells to obtain axons with a larger diameter. Western blotting and immunofluorescence studies were performed to confirm the neuronal status of the cells and diameter of the processes. In this study, we show that addition of cAMP-inducing factors to hNT2 cells resulted in rapid morphological changes including the development of processes with a larger diameter.

1-Methyl-3-isobutylxanthine↗
PubMed2006

Models of esophageal carcinogenesis.

Attempting to formulate a model for the development of esophageal cancer, we have undertaken three interrelated projects: (1) identification and characterization of stem cells as a basis to understand lineage specification in the normal esophageal squamous epithelium and possibly transdifferentiation into an intestinal metaplastic phenotype that defines Barrett's esophagus; (2) elucidation of the role of epidermal growth factor receptor and signaling mechanisms in proliferation and transformation in squamous cell carcinoma; and (3) modeling transformation in organotypic culture, a 3-dimensional culture system, as a platform for investigating epithelial-stromal interactions. It is hoped that development of these models for carcinogenesis will permit formulation of effective chemoprevention, diagnostic, and therapeutic strategies.

Animals↗
PubMed2003

Stem cell and niche development in the postnatal rat testis.

Adult tissue stem cells self-renew and differentiate in a way that exactly meets the biological demand of the dependent tissue. We evaluated spermatogonial stem cell (SSC) activity in the developing rat testis and the quality and accessibility of the stem cell niche in wild type, and two busulfan-treated models of rat pup recipient testes using an SSC transplantation technique as a functional assay. While our results revealed a 69-fold increase in stem cell activity during rat testis development from neonate to adult, only moderate changes in SSC concentration were observed, and stem cells from neonate, pup, and adult donor testes produce spermatogenic colonies of similar size. Analysis of the stem cell niche in recipient rat testes demonstrated that pup testes support high levels of donor stem cell engraftment when endogenous germ cells are removed or compromised by busulfan treatment. Fertility was established when rat pup donor testis cells were transplanted into fetal- or pup-busulfan-treated recipient rat pup testes, and the donor genotype was transmitted to subsequent generations. These results provide insight into stem cell/niche interactions in the rat testis and demonstrate that techniques originally developed in mice can be extended to other species for regenerative medicine and germline modification.

Animals↗
PubMed2003

In vitro differentiation of embryonic stem cells into mineralized osteoblasts.

Embryonic stem cells are pluripotent cells derived from the inner cell mass of mouse blastocysts that have been shown to differentiate spontaneously into cell types representing all three germ layers. This study shows that ES cells were induced to differentiate in vitro into mineralized osteoblasts under the influence of ascorbic acid, beta-glycerophosphate and 1alpha,25-OH vitamin D3. The activity of alkaline phosphatase, an early osteoblast marker, was found to be increased around day 12 of culture. Mineralized cells were clearly identified by histochemical staining, which detects mineralized calcium. The major noncollagenous component of bone matrix, osteocalcin, was localized to the mineralized cells by immunofluorescence. The expression of bone-specific genes was analyzed by real-time quantitative PCR. Osteocalcin and bone sialoprotein (BSP) were identified as early as in the fourth week of embryonic stem cell culture, both being characteristic for late stages of osteoblastic differentiation, indicating that at this time of culture the identified cells represent "mature" osteoblasts. The osteoblast-specific transcription factor Cbfa1 was induced a few days earlier. The expression of osteopontin and osteonectin, both being involved in binding calcium ions and hydroxyapatite during mineralization processes, as well as of collagen type I, representing by far the most predominant collagen in vertebrate organisms, is enhanced at the beginning of the second culture week upon addition of supplements. In the third week of culture, treated cells showed a second peak of osteopontin, osteonectin and collagen type I expression, osteopontin and osteonectin being stimulated 3-4-fold and collagen type I being induced 6-fold over control values. Alkaline phosphatase (ALP) expression was enhanced at the beginning of the third week of culture and was found to be increased again at later stages of culture at days 27-34. The in vitro differentiation of mouse embryonic stem cells into osteoblasts may provide a suitable model for studying the molecular processes of osteoblastic development in vivo.

Animals↗
PubMed1999

Oval cell numbers in human chronic liver diseases are directly related to disease severity.

The risk of developing hepatocellular carcinoma is significantly increased in patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C infection. The precise mechanisms underlying the development of hepatocellular carcinoma in these conditions are not well understood. Stem cells within the liver, termed oval cells, are involved in the pathogenesis of hepatocellular carcinoma in animal models and may be important in the development of hepatocellular carcinoma in human chronic liver diseases. The aims of this study were to determine whether oval cells could be detected in the liver of patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C, and whether there is a relationship between the severity of the liver disease and the number of oval cells. Oval cells were detected using histology and immunohistochemistry in liver biopsies from patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. Oval cells were not observed in normal liver controls. Oval cell numbers increased significantly with the progression of disease severity from mild to severe in each of the diseases studied. We conclude that oval cells are frequently found in subjects with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. There is an association between severity of liver disease and increase in the number of oval cells consistent with the hypothesis that oval cell proliferation is associated with increased risk for development of hepatocellular carcinoma in chronic liver disease.

Cell Count↗
PubMed1998

Natural killer and B-lymphoid potential in CD34+ cells derived from embryonic stem cells differentiated in the presence of vascular endothelial growth factor.

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels, differentiation intermediates were investigated. ES cells generated progeny expressing CD34, which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7, the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells, and such lymphoid potential was highly enriched in the CD34+ population. Interestingly, the cytotoxic cells expressed the natural killer (NK) cell markers, such as NKR-P1, perforin, and granzymes, classified into two types, one of which showed target specificity of NK cells. Thus, ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells, and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate, on which VEGF may play an important role.

Animals↗
PubMedDate not supplied

Proliferation and differentiation of mouse embryonic stem cells in APA microcapsule: A model for studying the interaction between stem cells and their niche.

Embryonic stem (ES) cells hold promise either as an in vitro model recapitulating early embryonic development or as a renewable source of therapeutically useful cells. Certain aspects of the microenvironment (or niche) play critical roles in determining the fate of ES cells. Here, we reported the feasibility of using the technique of microencapsulation to study the interaction between ES cells and their tissue niche. ES cells' growth, viability, and differentiation in vitro were evaluated when they were enclosed in solid or liquefied core APA microcapsules. In comparison with those microcapsules with solid cores, the liquefied capsules provided a more suitable culture environment for the growth of ES cells. In addition, behavior of encapsulated ES cells in vivo was observed after their being implanted into mouse peritoneal cavities. In contrast to the prolonged lag phase in vitro, ES cells encapsulated grew much faster in vivo. Typical markers for the undifferentiated ES cells, such as AP, SSEA-1, and Oct-4 gene, were also tracked by immunochemistry and RT-PCR. Results showed that expression of markers remained high over 2 weeks of culture in vitro. However, decreased expression of markers was found in those samples in vivo with time passage. These findings implied that it was the combination of the intrinsic characteristics of ES cells and their microenvironment that regulated their fate. The APA-ES cells system may provide an optimal model to study the interaction between stem cells and their tissue niches.

Alginates↗
PubMed2003

Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization.

In spite of the attention given to the study of mesenchymal stem cells (MSCs) derived from the bone marrow (BM) of humans and other species, there is a lack of information about murine MSCs. We describe the establishment of conditions for the in vitro expansion of plastic-adherent cells from murine BM for over 50 passages, and provide their characterization regarding morphology, surface marker profile and growth kinetics. These cells were shown to differentiate along osteogenic and adipogenic pathways, and to support the growth and differentiation of haematopoietic stem cells, and were thus operationally defined as murine mesenchymal stem cells (mMSCs). mMSCs were positive for the surface markers CD44, CD49e, CD29 and Sca-1, and exhibited a homogeneous, distinctive morphology. Their frequency in the BM of adult BALB/c and C57Bl/6 mice, normal or knockout for the alpha-L-iduronidase (IDUA) gene, was preliminarily estimated to be 1 per 11,300-27,000 nucleated cells. The emergence of a defined methodology for the culture of mMSCs, as well as a comprehensive understanding of their biology, will make the development of cellular and genetic therapy protocols in murine models possible, and provide new perspectives in the field of adult stem cells research.

Animals↗
PubMed2003

Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background.

Human neo-organ formation from stem cells can only be assayed by in vivo xenotransplantation. The human nonobese diabetic-severe combined immunodeficient (HuNOD/scid) CD34+ cell transplantation is a model that allows examination of hematopoietic tissue formation, although human hematopoietic cell maturation is abortive. Conventional humanization of the cytokine microenvironment has depended on generation of human cytokine-transgenic mice in strains appropriate for conventional plasmid microinjection, followed by backcrossing, a costly and time-consuming approach. Lentiviral vector infection of single-cell embryos was recently reported to produce transgenic animals. Using this approach, we have generated direct human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic mice from lentivirus-microinjected NOD/scid embryos, with 68% efficiency and 100% penetrance; this allowed us to obtain NOD/scid transgenic mice with considerable savings of resources. This powerful technique should assist in producing novel mouse models for the study of human blood cell lineage development and other human neo-organs from stem cell xenotransplantation for which a similar "humanization" rationale may be required.

Animals↗
PubMed2003

From embryos to embryoid bodies: generating blood from embryonic stem cells.

Differentiation of embryonic stem (ES) cells in vitro yields abundant hematopoietic progenitors, but achieving stable hematopoietic engraftment of irradiated mice has proven difficult, begging the question of whether ES cells give rise to hematopoietic stem cells in vitro, and limiting the application of ES cells as experimental and therapeutic models. We have employed a number of hematopoietic regulatory genes to probe the nature and developmental potential of ES-derived blood precursors. The chronic myeloid leukemia-associated BCR/ABL oncoprotein transforms a novel class of ES-derived embryonic hematopoietic stem cell that represents a common progenitor of primitive erythropoiesis and definitive lymphoid-myeloid blood development. Expression of the homeobox gene HoxB4 generated normal, non-leukemic hematopoietic progenitors that enabled long-term, multilineage hematopoietic engraftment in primary and secondary mouse recipients. We have used these repopulating hematopoietic stem cells to model therapeutic transplantation from ES cells. We treated an immunodeficient Rag2(-/-) mouse by therapeutic cloning, that is, isogenic ES cell generation by somatic cell nuclear transfer, gene correction, and cell replacement therapy. Comparable approaches with human ES cells are being developed to lay the foundation for cellular therapies in patients with a variety of bone marrow diseases.

Animals↗
PubMed2005

Regulation of hematopoiesis by retinoid signaling.

The discovery that retinoic acid efficiently stimulates the terminal differentiation of granulocytic leukemia cells had a major impact on clinical hematology, but has also inspired research into the normal function of the retinoid signaling pathway during hematopoiesis. New animal models and loss-of-function approaches have successfully revealed requirements for the pathway at defined embryonic stages that are relevant for distinct hematopoietic cell populations. For example, novel insight has been gained regarding the function of retinoids in yolk sac hematovascular development, fetal erythropoiesis, T-cell homing, and hematopoietic stem and progenitor cell biology. The lessons learned so far indicate that future development of sophisticated animal models will be needed to fully understand the intricacy and specificity of this complex signaling pathway, but that this effort will be productive and continue to inform both basic and clinical research on many fronts.

Animals↗