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At least 217 records · Page 12Linked to original sources
PubMed2005

Transgenic analysis of the stem cell leukemia +19 stem cell enhancer in adult and embryonic hematopoietic and endothelial cells.

Appropriate transcriptional regulation is critical for the biological functions of many key regulatory genes, including the stem cell leukemia (SCL) gene. As part of a systematic dissection of SCL transcriptional regulation, we have previously identified a 5,245-bp SCL +18/19 enhancer that targeted embryonic endothelium together with embryonic and adult hematopoietic progenitors and stem cells (HSCs). This enhancer is proving to be a powerful tool for manipulating hematopoietic progenitors and stem cells, but the design and interpretation of such transgenic studies require a detailed understanding of enhancer activity in vivo. In this study, we demonstrate that the +18/19 enhancer is active in mast cells, megakaryocytes, and adult endothelium. A 644-bp +19 core enhancer exhibited similar temporal and spatial activity to the 5,245-bp +18/19 fragment both during development and in adult mice. Unlike the +18/19 enhancer, the +19 core enhancer was only active in adult mice when linked to the eukaryotic reporter gene human placental alkaline phosphatase. Activity of a single core enhancer in HSCs, endothelium, mast cells, and megakaryocytes suggests possible overlaps in their respective transcriptional programs. Moreover, activity in a proportion of thymocytes and other SCL-negative cell types suggests the existence of a silencer elsewhere in the SCL locus.

Animals↗
PubMed2004

Making new beta cells from stem cells.

In 2000, Shapiro et al. provided compelling "proof of principle" data showing that the transplantation of human islets, purified from cadaveric material, could restore severely diabetic, Type 1 patients to insulin independence. This demonstration prompted renewed efforts to find an alternative and sustainable source of surrogate islet cells for cell therapy. Experiments involving adult ductal and liver "stem" cells, or embryonic stem cells, are prominent amongst these endeavors and are reviewed in this article. Whilst there are many published claims to success in converting ES cells into insulin secreting, glucose responsive cells, all require careful reinterpretation in the light of findings that cells can adsorb insulin present in growth media. It is likely that work with adult cells is less prone to this potential artifact and significant progress has been made in producing insulin-secreting cells. Assessment of in vivo function in the surrogate cells is most frequently made using cell transplantation into toxin-induced, diabetic mice, but this model is rarely used to maximal advantage. In many cases, it remains unclear whether reductions in the hyperglycemia result from insulin secretion from the transplanted cells or are due to recovery of endogenous islet function. In this latter context, experiments are reviewed where endogenous stimulation of recovery is engendered even by irradiated donor cells.

Animals↗
PubMed2006

Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood.

OBJECTIVE: Generation and expansion of pluripotent unrestricted somatic stem cells (USSCs) and mesenchymal cells (MSCs) from human cord blood as well their functional characterization were assessed. METHODS: USSC generation was initiated from fresh and cryopreserved cord blood (CB). Culture conditions and functional assays distinguishing between USSCs and MSCs (e.g., endodermal differentiation) are described. RESULTS: USSC cultures were initiated from 573 placental CB samples with a total generation frequency of 35.4% (n = 203), whereas successful generation of USSCs from cryopreserved products was scare. Medium, dexamethasone, and the fetal calf serum source are the major parameters to generate and expand the cells in a pluripotent state (osteogeneic, neural, and endodermal differentiation). CONCLUSIONS: USSC or MSC cultures can be easily obtained from fresh CB samples, good manufacturing practice production of such cells is feasible. These USSCs or MSCs from CB, when matched for HLA antigens, may serve as an allogeneic stem cell source for the development of cellular therapy for tissue repair.

Cell Culture Techniques↗
PubMed1993

New developments in stem cell detection and stem cell selection in the context of autologous transplantations.

The concept of bone marrow transplantation is now enlarged to hemopoietic stem cell transplantation. Basic research on hemopoiesis, its regulation and markers, allowed concrete progress in the field of autologous transplantation. We report two applications: 1) the improvement of methods for detection of stem cells, which ensure engraftment, using recombinant growth factors, allowed a more sensitive determination of chemotherapeutic in vitro treatments toxicity upon normal progenitor cells; 2) large scale selection of hemopoietic progenitor/stem cell using the CD 34 immunological marker: its application to autologous transplantation.

Bone Marrow Purging↗
PubMed1987

In vivo radiosensitivity and recovery pattern of the hematopoietic precursor cells and stem cells in mouse bone marrow.

Survival curves were determined for colony-forming hematopoietic stem cells (CFU-Mix and CFU-S10) as well as precursor cells (CFU-E, CFU-C, CFU-F, and BFU-E) in bone marrow of the mouse at various times up to 4 weeks after whole-body irradiation (1.5 or 3.0 Gy) in order to elucidate in vivo radiosensitivity and recovery patterns. These measurements represented the first attempts to examine CFU-Mix simultaneously with the other cell populations. CFU-E (D0 = 53 rad) and BFU-E (D0 = 68 rad) were the most radiosensitive. CFU-S10 (D0 = 81 rad) had intermediate radiosensitivity. CFU-Mix (D0 = 144 rad) and CFU-C (D0 = 157 rad) were relatively radioresistant. CFU-F (D0 = 257 rad) was the most radioresistant. The precursor and stem cells could be classified into three groups based on the recovery pattern. The first group, consisting of CFU-Mix, BFU-E, and CFU-S10, showed very slow recovery and did not reach normal levels even after day 28. CFU-E, the second group, showed the most severe depletion immediately after irradiation, and recovered most quickly with an overshoot at day 5. CFU-C and CFU-F cells, forming the third group, decreased more gradually and slightly, and recovered to the normal level after a transient rise by day 10-14.

Animals↗
PubMed2007

Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells.

OBJECTIVE: Human embryonic stem cells (hESCs) have been shown to generate CD34(+) primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73(+) multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34(+) hematopoietic cells and CD73(+) mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73(+) cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34(+) cells are generated. MATERIALS AND METHODS: We cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34(+) and CD73(+) cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34(+) cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73(+) cells generated using in vitro differentiation assays. RESULTS: We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34(+) hematopoietic and CD73(+) MSC generation follows a similar pattern. We sorted the CD34(+) cells and showed that they can generate hematopoietic progenitor colonies. Starting with cocultured cells on day 8, and through an enrichment procedure, we also could generate a pure population of MSCs. These hESC-derived MSCs had typical morphological and cell surface marker characteristics of adult bone marrow-derived MSCs, and could be differentiated toward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs. CONCLUSIONS: OP9 cells when cocultured with hESCs support simultaneous generation of CD34(+) primitive hematopoietic cells and CD73(+) MSCs from hESCs.

5'-Nucleotidase↗
PubMed1998

Autoimmune diseases as stem cell disorders: normal stem cell transplant for their treatment (Review).

Using various animal models for autoimmune diseases, we have found that autoimmune diseases are stem cell disorders. The transplantation of normal hemopoietic stem cells (HSCs) can be used to treat autoimmune diseases, whereas the transplantation of abnormal HSCs from autoimmune-prone mice to normal mice leads to the induction of autoimmune diseases in recipients. To elucidate the differences between normal and abnormal HSCs, we have established a new method for purifying pluripotent-HSCs (P-HSCs), and have compared the qualitative differences. Although normal P-HSCs cannot proliferate under major histocompatibility complex (MHC)-incompatible microenvironments (stromal cells), abnormal P-HSCs can proliferate under such conditions. In addition, the proliferation of abnormal P-HSCs is much faster than that of normal P-HSCs. These findings indicate that abnormal P-HSCs are more resilient than normal P-HSCs. Therefore, we propose that allogeneic bone marrow transplantation (BMT) (not autologous BMT) should be applied to the treatment of autoimmune diseases. human data on BMT in autoimmune diseases is reviewed, and the conditions essential for successful BMT, including tolerance induction, are discussed.

Animals↗
PubMedDate not supplied

Biomimetic three-dimensional cultures significantly increase hematopoietic differentiation efficacy of embryonic stem cells.

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation, we cultured mouse ES cells on 3D, highly porous, biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however, unlike 2D differentiation, these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly, the efficiency of hematopoietic precursor cell (HPC) generation on 3D, as indicated by the expression of various HPC-specific surface markers (CD34, Sca-1, Flk-1, and c-Kit) and colony-forming cell (CFC) assays, was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs.

Animals↗
PubMed1997

Marrow stromal cells as stem cells for nonhematopoietic tissues.

Marrow stromal cells can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic. The cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Therefore, marrow stromal cells present an intriguing model for examining the differentiation of stem cells. Also, they have several characteristics that make them potentially useful for cell and gene therapy.

Adipocytes↗
PubMed1998

Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype.

BACKGROUND: In this study we tried to achieve successful transplantation in patients with acute leukemia with the use of hematopoietic stem cells from donors who shared only one HLA haplotype with the recipient (a "full-haplotype mismatch"). To prevent graft failure, large doses of T-cell-depleted hematopoietic stem cells were transplanted after a conditioning regimen of enhanced myeloablation and immunosuppression was administered to the recipient. METHODS: Forty-three patients with high-risk acute leukemia who were scheduled for transplantation received total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. The graft consisted of peripheral-blood progenitor cells that had been mobilized in the donor with recombinant granulocyte colony-stimulating factor and also, in 28 cases, bone marrow. Bone marrow from the donor was depleted of T lymphocytes by processing with soybean agglutinin and E-rosetting. T-cell depletion of peripheral-blood mononuclear cells was achieved by E-rosetting followed by positive selection of CD34+ cells. No post-transplantation prophylaxis against graft-versus-host disease (GVHD) was administered. RESULTS: In all the patients, full donor-type engraftment was achieved. In none of the patients who could be evaluated did acute or chronic GVHD develop. Regimen-related toxicity was minimal. Eleven of the 23 patients with acute lymphoblastic leukemia had a relapse, as did 2 of the 20 patients with acute myeloid leukemia. Transplantation-related mortality was 40 percent. After a median follow-up of 18 months (range, 8 to 30), 12 of the 43 patients were alive and free of disease. All surviving patients had a good quality of life. CONCLUSIONS: The main limitations of transplantation of bone marrow from donors who are matched with the recipient for only one HLA haplotype GVHD and graft failure - can be overcome. Since most patients have a relative with one haplotype mismatch, advances in this method will increase the availability of hematopoietic-cell transplantation as curative therapy for acute leukemia.

Adolescent↗
PubMed2003

Mesenchymal stem cells and hematopoietic stem cell transplantation.

Techniques have recently beome available to isolate and grow mesenchymal progenitors and to manipulate their growth under defined in vitro culture conditions. As a result mesenchymal stem cells can be rapidly expanded to numbers that are required for clinical application. This has allowed the clinical testing of culture-expanded MSCs in the context of hematopoietic stem cell transplantation. In this paper we discuss the role of MSCs in hematopoietic engraftment after transplantation.

Animals↗
PubMed1996

[High-dose therapy of cancer with CD34 positive cells as stem cell support].

In this article we report our initial clinical experiences in connection with immunomagnetic isolated CD34-positive cells from peripheral blood progenitor cells. Six patients, five with breast cancer and one with non-Hodgkin's lymphoma, were mobilized by chemotherapy and G-CSF (5ug/kg). CD34-positive cells were isolated by means of immunomagnetic beads (Dynalbeads) and Isolex 300 Cell Separator (Baxter, USA). Mean purity of isolated CD34-positive cells was 97% (94.7-99.7) and mean yield was 54% (35-68). Three patients were treated with high dose therapy followed by reinfusion of CD34-positive cells as stem cell support. Recovery of neutrophils (> 0.5 x 10(9) leucocytes/liter) occurred at day 8, 11 and 13 and of platelets (> 20 x 10(9) platelets/litre) at day 9,14 and 32. It is concluded that immunomagnetic isolated CD34-positive cells give high purity and yield. Although use of CD34-positive cells reduces the content of contaminating tumour cells in the graft, breast cancer cells were still detectable in two out of five CD34-positive cell products.

Antigens, CD34↗
PubMed2006

High passage number of stem cells adversely affects stem cell activation and myocardial protection.

Progenitor cell plasticity enhances positive remodeling of damaged tissue. We and others have previously shown that progenitor cells may limit apoptosis and modulate inflammation in part by the production of growth factors. However, recent studies suggest that progenitor cells senesce and lose their differentiation potential with increasing time in culture and passage. We hypothesize that murine bone marrow mesenchymal stem cells (MSCs) are cardioprotective against ischemia/reperfusion injury in the isolated perfused rat heart, and that passage number has an adverse effect on MSC activation and cardioprotection. Adult male and female Sprague-Dawley rat hearts were isolated, perfused via Langendorff model, and subjected to ischemia/reperfusion. Mouse MSCs were harvested, cultured, suspended in perfusate, and infused before global index ischemia. Hearts were assigned to controls or infusion with passage 3, 5, or 10 MSCs. In addition, MSCs in culture were stressed by hypoxia and increasing doses of endotoxin (lipopolysaccharide). Mesenchymal stem cell activation was determined by measuring vascular endothelial growth factor production with enzyme-linked immunosorbent assay. All data are reported as mean +/- SEM and were analyzed with 2-way analysis of variance. Differences are considered significant if P < 0.05. Passage 3 murine MSC infusion in hearts before ischemia reduced the depression of left ventricular developed pressure, attenuated the increase of end-diastolic pressure, and reduced the depression of +dP/dT and -dP/dT. However, the MSC protective effect disappeared in hearts infused with passage 5 and passage 10 MSCs. Although hypoxia and lipopolysaccharide resulted in significant activation of MSCs, passage 3 MSCs demonstrated significantly greater vascular endothelial growth factor release than passage 5 and 10 MSCs. Acute murine MSC infusion confers protection in isolated rat hearts. However, high passage number has an adverse effect on MSC activation and protection. This portends limited ex vivo expansion before possible therapeutic use.

Animals↗
PubMed2000

Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell-depleted stem cell transplantation.

We evaluated the efficacy, toxicity, and outcome of preemptive ganciclovir (GCV) therapy in 80 cytomegalovirus (CMV)-seropositive patients allografted between 1991 and 1996 and compared their outcome to 35 seronegative patients allografted during the same period. Both cohorts were comparable with respect to diagnosis and distribution of high- versus standard-risk patients. All patients received a stem cell graft from an HLA-identical sibling donor, and grafts were partially depleted of T cells in 109 patients. Patients were monitored for CMV antigenemia by leukocyte expression of the CMV-pp65 antigen. Fifty-two periods of CMV reactivation occurring in 30 patients were treated preemptively with GCV. A favorable response was observed in 48 of 50 periods, and only 2 patients developed CMV disease: 1 with esophagitis and 1 with pneumonia. Ten of 30 treated patients developed GCV-related neutropenia (less than 0.5 x 10(9)/L), which was associated with a high bilirubin at the start of GCV therapy. Overall survival at 5 years was 64% in the CMV-seronegative cohort and 40% in the CMV-seropositive cohort (P =.01). Increased treatment-related mortality accounted for inferior survival. CMV seropositivity proved an independent risk factor for developing acute graft-versus-host disease, and acute graft-versus-host disease predicted for higher treatment-related mortality and worse overall survival in a time-dependent analysis. We conclude that, although CMV disease can effectively be prevented by preemptive GCV therapy, CMV seropositivity remains a strong adverse risk factor for survival following partial T-cell-depleted allogeneic stem cell transplantation.

Adolescent↗
PubMed1997

Embryonic stem cells and hematopoietic stem cell biology.

Two advances in murine embryonic stem (ES) cell technology and their applications for the study of hematopoietic stem cells (HSCs) are discussed in this article. First, ES cells induced to differentiate in vitro form hematopoietic lineages in a fashion that recapitulates the ontogeny of blood formation in the embryo. This system offers a unique opportunity to isolate, examine, and manipulate the most primitive hematopoietic progenitors. Second, targeted gene ablation (knockout) studies in ES cells have identified several genes that are required for normal hematopoiesis and may function in the formation, maintenance, and differentiation of HSCs. Insights into murine hematopoiesis gained through the study of ES cells generally should be applicable to other vertebrates, including humans.

Animals↗
PubMed2000

Retroviral expression in embryonic stem cells and hematopoietic stem cells.

Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Adoptive Transfer↗
PubMed2002

Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell-depleted stem cell transplantation.

We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence.

Antigens, Viral↗
PubMed2000

Cell biology. Stem cell scorecard.

As researchers continue to explore the potential uses of stem cells obtained from a variety of sources (see main text), governments around the world are grappling with whether to allow research on stem cells derived from human embryos. Governments are cautious yet increasingly open to the new research, which may eventually yield treatments for a variety of diseases from Parkinson's to diabetes.

Australia↗