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PubMed2004

Can the life span of human marrow stromal cells be prolonged by bmi-1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation?

BACKGROUND: Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell-cycle-associated genes can differentiate into cardiomyocytes in vitro. METHODS: We attempted to prolong the life span of hMSCs by infecting retrovirus encoding bmi-1, human papillomavirus E6 and E7, and/or human telomerase reverse transcriptase genes. To determine whether the hMSCs with an extended life span could differentiate into cardiomyocytes, 5-azacytidine-treated hMSCs were co-cultured with fetal cardiomyocytes in vitro. RESULT: The established hMSCs proliferated over 150 population doublings. On day 3 of co-cultivation, the hMSCs became elongated, like myotubes, began spontaneously beating, and acquired automaticity. Their rhythm clearly differed from that of the surrounding fetal mouse cardiomyocytes. The number of beating cardiomyocytes increased until 3 weeks. hMSCs clearly exhibited differentiated cardiomyocyte phenotypes in vitro as revealed by immunocytochemistry, RT-PCR, and action potential recording. CONCLUSIONS: The life span of hMSCs was prolonged without interfering with cardiomyogenic differentiation. hMSCs with an extended life span can be used to produce a good experimental model of cardiac cell transplantation and may serve as a highly useful cell source for cardiomyocytic transplantation.

Action Potentials↗
PubMed2002

Early progression from dimethyl sulfoxide-induced G(0)/G(1) arrest in L(1210) cells.

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G(0)/G(1) arrest in mouse leukemia L(1210) cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA-synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36 h. In conclusion, mouse leukemia L(1210) cells, which were treated with 1.5% DMSO for 96 h, tolerated the treatment and reversed the cell cycle arrest within 36 h.

Animals↗
PubMed2003

Barrett's esophagus: environmental influences in the progression of dysplasia.

Barrett's esophagus (BE) is composed of multiple lineages including Paneth cells and endocrine cells in addition to gastric and intestinal cells. Although the origin of the BE stem cell is a matter of conjecture, the stem cells are clearly multipotent, and therefore the phenotype is restricted by genomic imprinting (termed restricted potency). Recent evidence suggests that the microenvironment may select various lineages. In this regard the proportion of gastric and specialized intestinal metaplastic cells has been attributed to the composition of the refluxate, acid or bile, respectively. Experimental evidence also implicates specific xenobiotics in this process, including bile acids. In particular we discuss the potential biologic roles of bile acids in epithelial adaptation from in vivo and in vitro models.

Adaptation, Physiological↗
PubMed2005

Effects produced by Royal Jelly on haematopoiesis: relation with host resistance against Ehrlich ascites tumour challenge.

Royal jelly (RJ) was shown to exhibit immunomodulatory properties, although its biological activity is still unclear. In order to elucidate the mechanism whereby RJ activates the immunological system, we examined the role of this substance on the haematopoietic response of Ehrlich ascites tumour (EAT)-bearing mice. Our results demonstrated that RJ prevented the myelosupression induced by the temporal evolution of the tumour and abrogated the splenic haematopoiesis observed in EAT-bearing mice. The stimulating effect of RJ was also observed in vitro on the multipotent bone marrow stem cells, evaluated by the long-term bone marrow cultures (LTBMCs). The study of survival clearly showed the antitumour activity of RJ. Treatment was given prophylactically for 20 days and therapeutically for 3, 8 and 13 days. Except for the treatment with the lower dose of 500 mg/kg, given for 23 days, all the other dose schedules were able to prolong survival. A more effective antitumoural response was observed with the more prolonged treatment regimen. In this regard, the administration of RJ for 33 days produced the highest protection reaching an extension of survival at about 38%, 71% and 85% for the doses of 500, 1000 and 1500 mg/kg, respectively, whereas with the 23 and 28 days treatment schedules, survival increased at a rate of 19% and 23%, respectively, and comparable results were found among the effective doses of RJ. Increased survival rate might be related to the decreased Prostaglandin E2 (PGE2) levels observed in EAT-bearing mice after RJ treatment. These results point to RJ as a promising modifier of biological response leading to myeloprotection and antitumour activity.

Adjuvants, Immunologic↗
PubMed2006

Decreased CD90 expression in human mesenchymal stem cells by applying mechanical stimulation.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent cells which can differentiate along osteogenic, chondrogenic, and adipogenic lineages. The present study was designed to investigate the influence of mechanical force as a specific physiological stress on the differentiation of (MSC) to osteoblast-like cells. METHODS: Human MSC were cultured in osteoinductive medium with or without cyclic uniaxial mechanical stimulation (2000 mustrain, 200 cycles per day, 1 Hz). Cultured cells were analysed for expression of collagen type I, osteocalcin, osteonectin, and CD90. To evaluate the biomineral formation the content of bound calcium in the cultures was determined. RESULTS: After 14 days in culture immunfluorescence staining revealed enhancement of collagen type I and osteonectin expression in response to mechanical stimulation. In contrast, mechanically stimulated cultures stained negative for CD90. In stimulated and unstimulated cultures an increase in the calcium content over time was observed. After 21 days in culture the calcium content in mechanical stimulated cultures was significantly higher compared to unstimulated control cultures. CONCLUSION: These results demonstrate the influence of mechanical force on the differentiation of human MSC into osteoblast-like cells in vitro. While significant enhancement of the biomineral formation by mechanical stimulation is not detected before 21 days, effects on the extracellular matrix became already obvious after 14 days. The decrease of CD90 expression in mechanically stimulated cultures compared to unstimulated control cultures suggests that CD90 is only transiently expressed expression during the differentiation of MSC to osteoblast-like cells in culture.

Journal Article↗
PubMed2004

Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain.

In many species, the Sox2 transcription factor is a marker of the nervous system from the beginning of its development, and we have previously shown that Sox2 is expressed in embryonic neural stem cells. It is also expressed in, and is essential for, totipotent inner cell mass stem cells and other multipotent cell lineages, and its ablation causes early embryonic lethality. To investigate the role of Sox2 in the nervous system, we generated different mouse mutant alleles: a null allele (Sox2beta-geo 'knock-in'), and a regulatory mutant allele (Sox2DeltaENH), in which a neural cell-specific enhancer is deleted. Sox2 is expressed in embryonic early neural precursors of the ventricular zone and, in the adult, in ependyma (a descendant of the ventricular zone). It is also expressed in the vast majority of dividing precursors in the neurogenic regions, and in a small proportion of differentiated neurones, particularly in the thalamus, striatum and septum. Compound Sox2(beta-geo/DeltaENH) heterozygotes show important cerebral malformations, with parenchymal loss and ventricle enlargement, and L-dopa-rescuable circling behaviour and epilepsy. We observed striking abnormalities in neurones; degeneration and cytoplasmic protein aggregates, a feature common to diverse human neurodegenerative diseases, are observed in thalamus, striatum and septum. Furthermore, ependymal cells show ciliary loss and pathological lipid inclusions. Finally, precursor cell proliferation and the generation of new neurones in adult neurogenic regions are greatly decreased, and GFAP/nestin-positive hippocampal cells, which include the earliest neurogenic precursors, are strikingly diminished. These findings highlight a crucial and unexpected role for Sox2 in the maintenance of neurones in selected brain areas, and suggest a contribution of neural cell proliferative defects to the pathological phenotype.

Alleles↗
PubMed2000

Fate restriction in limb muscle precursor cells precedes high-level expression of MyoD family member genes.

The mechanisms by which pluripotent embryonic cells generate unipotent tissue progenitor cells during development are unknown. Molecular/genetic experiments in cultured cells have led to the hypothesis that the product of a single member of the MyoD gene family (MDF) is necessary and sufficient to establish the positive aspects of the determined state of myogenic precursor cells: i.e., the ability to initiate and maintain the differentiated state (Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S. et al. (1991) Science 251, 761-766). Embryonic cell type determination also involves negative regulation, such as the restriction of developmental potential for alternative cell types, that is not directly addressed by the MDF model. In the experiments reported here, phenotypic restriction in myogenic precursor cells is assayed by an in vivo 'notochord challenge' to evaluate their potential to 'choose' between two alternative cell fate endpoints: cartilage and muscle (Williams, B. A. and Ordahl, C. P. (1997) Development 124, 4983-4997). Two separate myogenic precursor cell populations were found to be phenotypically restricted while expressing the Pax3 gene and prior to MDF gene activation. Therefore, while MDF family members act positively during myogenic differentiation, phenotypic restriction, the negative aspect of cell specification, requires cellular and molecular events and interactions that precede MDF expression in myogenic precursor cells. The qualities of muscle formed by the determined myogenic precursor cells in these experiments further indicate that their developmental potential is intermediate between that of myoblastic stem cells taken from fetal or adult tissue (which lack mitotic and morphogenetic potential when tested in vivo) and embryonic stem cells (which are multipotent). We hypothesize that such embryonic myogenic progenitor cells represent a distinct class of determined embryonic cell, one that is responsible for both tissue growth and tissue morphogenesis.

Animals↗
PubMed1986

Distribution of developmental markers in rat mammary tumors induced by N-nitrosomethylurea.

We have examined the distribution of immunological markers in 55 intraductal carcinomas induced in rats by N-nitrosomethylurea using several different regimens of carcinogen treatment. The goal was to determine the possible relationship of the marker distribution to those existing at various stages of development of the mammary gland. We have also examined two passage lines in syngeneic rats derived from two of the tumors. The distribution of markers was not affected by the regimen of administration. The primary tumors were found to maintain the general topography of mammary ducts but with infoldings of the basal layer without accompanying stroma. We attribute this to an abnormal expression of the tendency of basal cells to migrate towards the lumen to generate luminal cells. The distribution of markers in the tumors was evaluated by identifying 13 special features of distribution that are common in tumors, using all-or-none criteria. The distribution of these features bears out the high heterogeneity of tumors in which the various features vary in a seemingly independent way. There is also heterogeneity within tumors, adjacent nodes often having different marker distributions. The distribution of the markers is related to that found in the early stages of mammary development. Because of this characteristic and of the fact that the tumors contain both basal and luminal cells, they must originate from multipotent cells, probably the stem cells present in end buds and ducts as already proposed by other work. As they develop, the tumors can both differentiate towards the adult type and dedifferentiate towards a fetal type. Of the two transplanted lines one retained the same general features of primary tumors in several passages, whereas the other evolved into a fusiform cell type with a marker distribution not seen at any stage of mammary development. Foci of similar cells were already present in the primary tumor, suggesting that the tendency to progress was already determined at an early stage. The fusiform cells are similar and probably equivalent to the fusiform cells that arise in vitro in cultures of rat mammary cancers.

Animals↗
PubMed2005

Stem cell-based therapy for Parkinson's disease.

Motor dysfunctions in Parkinson's disease are considered to be primarily due to the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Pharmacological therapies based on the principle of dopamine replacement are extremely valuable, but suffer from two main drawbacks: troubling side effects (e.g. dyskinesia) and loss of efficacy with disease progression. Transplantation of embryonic dopaminergic neurons has emerged as a therapeutic alternative. Enthusiasm following the success of the initial open-label trials has been dampened by the negative outcome of double-blind placebo controlled trials. Additionally, the emergence of graft-related dyskinesia indicates that the experimental grafting procedure requires further refinement before it can be developed into a therapy. Shortage of embryonic donor tissue limits large-scale clinical transplantation trials. We review three of the most attractive tissue sources of dopaminergic neurons for cell replacement therapy: human embryonic ventral mesencephalic tissue, embryonic and adult multipotent region-specific stem cells and embryonic stem cells. Recent developments in embryonic stem cell research and on their implications for a future transplantation therapy in Parkinson's disease are described. Finally, we discuss how human embryonic stem cells can be differentiated into dopaminergic neurons, and issues such as the numbers of dopaminergic neurons required for success and the risk for teratoma formation after implantation.

Animals↗
PubMed1999

The myeloid-lymphoid initiating cell (ML-IC) assay assesses the fate of multipotent human progenitors in vitro.

Hematopoietic stem cells (HSC) are cells with self-renewing multilineage differentiation potential. Although engraftment in xenogeneic recipients can be used to measure human HSC, these assays do not allow assessment of individual progenitors. We developed an in vitro assay that allows the identification of a single human bone marrow progenitor closely related to HSC, which we termed "Myeloid-Lymphoid Initiating Cell," or ML-IC, because it is capable of generating multiple secondary progenitors that can reinitiate long-term myeloid and lymphoid hematopoiesis in vitro. The assay is done in contact with murine AFT024 fetal liver stromal cells and with Flt3-Ligand, stem cell factor, and interleukin-7. In this assay, 0.2% to 1.7% of Lin -/34(+)/DRdim cells could generate 1 to 3 long-term culture initiating cells (LTC-IC) as well as 1 to 4 NK-IC after 4 to 6 weeks. In addition, this assay measures contribution of net-progenitor conservation and net-progenitor proliferation over time, providing insight in the fate of individual LTC-IC and NK-IC. This assay will prove useful to enumerate the number of very primitive human progenitors with multilineage differentiation potential, as well as to evaluate future ex vivo culture conditions.

Animals↗
PubMed2006

Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.

The recent identification of "side population" (SP) cells in a number of unrelated human cancers and their normal tissue sources has renewed interest in the hypothesis that cancers may arise from somatic stem/progenitor cells. The high incidence of recurrence attributable to multidrug resistance and the multiple histologic phenotypes indicative of multipotency suggests a stem cell-like etiology of ovarian cancer. Here we identify and characterize SP cells from two distinct genetically engineered mouse ovarian cancer cell lines. Differential efflux of the DNA-binding dye Hoechst 33342 from these cell lines defined a human breast cancer-resistance protein 1-expressing, verapamil-sensitive SP of candidate cancer stem cells. In vivo, mouse SP cells formed measurable tumors sooner than non-SP (NSP) cells when equal numbers were injected into the dorsal fat pad of nude mice. The presence of Mullerian Inhibiting Substance (MIS) signaling pathway transduction molecules in both SP and NSP mouse cells led us to investigate the efficacy of MIS against these populations in comparison with traditional chemotherapies. MIS inhibited the proliferation of both SP and NSP cells, whereas the lipophilic chemotherapeutic agent doxorubicin more significantly inhibited the NSP cells. Finally, we identified breast cancer-resistance protein 1-expressing verapamil-sensitive SPs in three of four human ovarian cancer cell lines and four of six patient primary ascites cells. In the future, individualized therapy must incorporate analysis of the stem cell-like subpopulation of ovarian cancer cells when designing therapeutic strategies for ovarian cancer patients.

Animals↗
PubMed1991

[Constitutional hematologic diseases. Perspectives of gene therapy].

Technical advances in molecular genetics have succeeded in elucidating the molecular basis of many hereditary diseases. The characterization of these abnormalities is currently giving rise to the concept of pharmacological treatment or, if possible, organ replacement (bone marrow grafting or liver transplantation). Genotherapy aims at replacing a single deficient gene by a functional gene introduced into an autologous, and therefore unrejectable, tissue. The haematopoietic stem cells are excellent targets for gene transfer, since the procurement, ex vivo, manipulation and reimplantation of these cells are easily performed. Several constitutional blood diseases would benefit from a causative treatment if a group of truly multipotent, self-renewing stem cells could integrate a transgene compensating for the hereditary deficit. The problems inherent in gene transfer, targeting on haematopoietic stem cells, mode of introduction of the transgene and its expression at a sufficiently high level are presented. Important progress has recently been made in this fields, and therapeutic applications to man can now be envisaged, although not all obstacles have been overcome. This concerns, in particular, the globin deficiency gene in beta thalassemia.

Adenosine Deaminase↗
PubMed2001

Stem cell bioengineering.

Tissue engineering and cellular therapies, either on their own or in combination with therapeutic gene delivery, have the potential to significantly impact medicine. Implementation of technologies based on these approaches requires a readily available source of cells for the generation of cells and tissues outside a living body. Because of their unique capacity to regenerate functional tissue for the lifetime of an organism, stem cells are an attractive "raw material" for multiple biotechnological applications. By definition they are self-renewing because on cell division they can generate daughter stem cells. They are also multipotent because they can differentiate into numerous specialized, functional cells. Recent findings have shown that stem cells exist in most, if not all, tissues, and that stem cell tissue specificity may be more flexible than originally thought. Although the potential for producing novel cell-based products from stem cells is large, currently there are no effective technologically relevant methodologies for culturing stem cells outside the body, or for reproducibly stimulating them to differentiate into functional cells. A mechanistic understanding of the parameters important in the control of stem cell self-renewal and lineage commitment is thus necessary to guide the development of bioprocesses for the ex vivo culture of stem cells and their derivates.

Adult↗
PubMed2006

Cluster analysis and gene expression profiles: a cDNA microarray system-based comparison between human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for tissue engineering cell therapy.

We investigated gene expression patterns and functional classifications regarding the clusters of human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs)--which possess a multipotent ability--because little is known about the precise moleculobiological clues by which these cells activate their differentiating ability or functionality to eventually form dentin and bone, respectively. We first verified the expressions of the alkaline phosphatase (ALP) gene, dentin matrix protein 1 (DMP-1), and dentinsialophosphoprotein (DSPP) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and consequently discovered the high expressions of these genes. Total RNA was also followed by hybridization with a human microarray system consisting of 12,814 genes. Analyses of gene expression patterns indicated several genes which encode extracellular matrix components, cell adhesion molecules, growth factors, and transcription regulators. Functional and clustering analyses of differences in gene expression levels revealed cell signaling, cell communication, or cell metabolism. In the future, information on the gene expression patterns of hDPSCs and hMSCs might be useful in determining the detailed functional roles of the relevant genes and applicable to stem cell therapies, and these cells could also be used as multipotent cell sources for gene technology and tissue engineering technology.

Adult↗
PubMed2006

Differential effect of leukaemogenic tyrosine kinases on cell motility is governed by subcellular localisation.

The chemokine, stromal cell-derived factor-1 (SDF-1) is a crucial regulator of stem cell homing and tethering, and potentiation of this pathway in leukaemias may contribute to the pathogenesis of the disease. A key second messenger in SDF-1 signal/response coupling is phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]. SDF-1 elevated PtdIns(3,4,5)P3 levels markedly in the multipotent FDCP-mix stem cell line. Similarly, transfection with BCR/ABL or TEL/PDGFRbeta leukaemogenic tyrosine kinases chronically elevated PtdIns(3,4,5)P3 levels. However, whilst an SDF-1 chemotactic response was observed in TEL/PDGFRbeta-transfected cells, in BCR/ABL cells this was markedly decreased, which was not due to Ras-pathway activation. Thus, multipotent cells can respond to SDF-1, despite chronic increases in this second messenger indicating that a discrete pool of SDF-1-stimulated PtdIns(3,4,5)P3 production drives the chemotactic response. To discern the mechanism for the differential effects of these oncogenes we considered subcellular localisation. As TEL/PDGFRbeta has a cytosolic location whilst BCR/ABL associates with actin, we removed the actin-binding domain from BCR/ABL. We observed relocation of BCR/ABL to the cytosol and increased SDF-1 responses. We conclude that the localisation of BCR/ABL to the cytoskeleton is essential for effects on motility and moderating SDF-1 responses is not essential in tyrosine kinase-mediated leukaemic transformation.

Actins↗
PubMed1997

Identification of a lineage of multipotent hematopoietic progenitors.

All multipotent hematopoietic progenitors in C57BL-Thy-1.1 bone marrow are divided among three subpopulations of Thy-1.1(lo) Sca-1+ Lin(-/lo) c-kit+ cells: long-term reconstituting Mac-1- CD4- c-kit+ cells and transiently reconstituting Mac-1(lo) CD4- or Mac-1(lo) CD4(lo) cells. This study shows that the same populations, with similar functional activities, exist in mice whose hematopoietic systems were reconstituted by hematopoietic stem cells after lethal irradiation. We demonstrate that these populations form a lineage of multipotent progenitors from long-term self-renewing stem cells to the most mature multipotent progenitor population. In reconstituted mice, Mac-1- CD4- c-kit+ cells gave rise to Mac-1(lo) CD4- cells, which gave rise to Mac-1(lo) CD4(lo) cells. Mac-1- CD4- c-kit+ cells had long-term self-renewal potential, with each cell being capable of giving rise to more than 10(4) functionally similar Mac-1- CD4- c-kit+ cells. At least half of Mac-1(lo) CD4- cells had transient self-renewal potential, detected in the spleen 7 days after reconstitution. Mac-1(lo) CD4(lo) cells did not have detectable self-renewal potential. The identification of a lineage of multipotent progenitors provides an important tool for identifying genes that regulate self-renewal and lineage commitment.

Animals↗
PubMed1998

In vitro reconstitution of human B-cell ontogeny: from CD34(+) multipotent progenitors to Ig-secreting cells.

We describe a long-term, in vitro culture system initiated with CD34(+) or CD34(+)CD38(-) umbilical cord blood hematopoietic progenitors that supports normal human B-lineage development, including the production of mature Ig-secreting B cells. In the first stage (human B-progenitor long-term culture [HB-LTC]), CD34(+) hematopoietic progenitors are cultured on the murine stromal cell line, S17, leading to the sustained production of large numbers of CD10(+), CD19(+) early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrogate light chain), cytoplasmic mu chain, and surface IgM expression were used to characterize the CD19(+) B progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B cells, and IgM+, IgD-/+ immature B cells. The limited expansion of IgM+ B cells and the immature surface phenotype of this population (IgM+, IgD+, CD10(+), CD38(+)) suggested that HB-LTC conditions were unable to provide appropriate signals for further differentiation. A second culture stage was used to determine if these immature B cells were functionally competent. Purified CD19(+) cells were transferred onto fibroblasts expressing human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell proliferation, modulation of the IgM+ cell surface phenotype to one consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated using two-stage cultures established with highly purified multipotent CD34(+)CD38(-) hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transition points in human B ontogeny, beginning with commitment to the B lineage and leading to development and activation of mature B cells.

Animals↗
PubMed2006

Contrasting mechanisms of stem cell maintenance in Drosophila.

Stem cells are self-renewing multipotent cells essential for development or homeostasis of many tissues. Stem cell populations can be found in most multicellular plants and animals. The mechanisms by which these populations are maintained are diverse, utilizing both intrinsic and extrinsic factors to regulate cell division and differentiation. The genetic tools of the fruitfly, Drosophila melanogaster, have permitted detailed characterization of two stem cell populations. In this review, we will examine these contrasting stem cell model systems from Drosophila and their relevance to stem cell populations in other organisms.

Animals↗