Inhibitor Of Stem Cell Proliferation Research Articles

Non-ammoniagenic proliferation and differentiation media for cultivated adipose tissue.

Ammonia (Amm), and its aqueous solved state, ammonium, which is produced from glutamine (Gln) metabolism, is a known inhibitor of stem cell proliferation in vitro. In the context of cultivated beef, primary bovine fibro-adipogenic progenitor cells (FAPs) need to be grown and differentiated for several weeks in vitro for the production of cultivated fat. In this study, the ammonium sensitivity of these cells was investigated by introducing ammonium chloride, which was found to inhibit their proliferation when above 5mM and their adipogenic differentiation when above 2mM. Novel serum-free proliferation and differentiation media were hence developed with the aim to suppress Amm production during expansion and adipogenesis. Glutamine substitutes, such as a-ketoglutarate (aKG), glutamate (Glt) and pyruvate (Pyr) were investigated. It was found that aKG based proliferation medium (PM) was the most effective in promoting and maintaining FAPs growth over several passages while the specific Amm production rate was reduced more than 5-fold. In terms of differentiation capacity, the substitution of glucose (Gluc) and Gln with galactose (Gal) and Pyr was shown to be the most effective in promoting FAPs differentiation into mature adipocytes, resulting in over 2-fold increase of fat volume per cell, while suppressing Amm production. Our findings suggest that FAPs do not require Gln as an essential nutrient but, on the contrary, possess all the necessary metabolic pathways to proliferate and subsequently differentiate in a Gln-free medium, resulting in decreased Amm production rates and seemingly synthesising glutamine de novo. These findings are important for prolonging the lifespan of culture medium, allowing for reduced costs and process interventions.

Olig2-Regulated Lineage-Restricted Pathway Controls Replication Competence in Neural Stem Cells and Malignant Glioma

Recent studies have identified stem cells in brain cancer. However, their relationship to normal CNS progenitors, including dependence on common lineage-restricted pathways, is unclear. We observe expression of the CNS-restricted transcription factor, OLIG2, in human glioma stem and progenitor cells reminiscent of type C transit-amplifying cells in germinal zones of the adult brain. Olig2 function is required for proliferation of neural progenitors and for glioma formation in a genetically relevant murine model. Moreover, we show p21(WAF1/CIP1), a tumor suppressor and inhibitor of stem cell proliferation, is directly repressed by OLIG2 in neural progenitors and gliomas. Our findings identify an Olig2-regulated lineage-restricted pathway critical for proliferation of normal and tumorigenic CNS stem cells.

BB-10010/MIP-1 alpha in vivo maintains haemopoietic recovery following repeated cycles of sublethal irradiation.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is an inhibitor of stem cell proliferation affording protection against damage from agents that express their cytotoxicity specifically in the DNA synthesis phase of the cell cycle. Its ability also to modify the self-renewal capacity of the regenerating cells is now shown to improve and maintain haemopoietic recovery following therapy (sublethal irradiation) whose cytotoxic damage is not limited solely to the DNA-S phase of this cycle. Such non-cell cycle-active cytotoxic agents are used clinically in repeated treatment regimens, which are often limited or terminated because of accumulating haemopoietic damage. BB-10010, a non-aggregating variant of MIP-1 alpha, was administered as a continuous dose (1600 micrograms kg-1 24 h-1) via a subcutaneously implanted pump over a period of 7 days. A dose of 4.5 Gy total-body gamma-rays was given 3-4 h after implantation. Day 8 and 12 spleen colony-forming units (CFU-S) were assayed on days 1, 7 and 14 after irradiation. This cycle of treatment was repeated four times (total 56 days), and on day 14 of the last two cycles the marrow-repopulating ability (MRA) was also measured. In the control bone marrow (no BB-10010) CFU-S fell to < 1% of normal within 1 day of irradiation and recovered to 40% at 14 days. Repeated treatments increased the level of damage, and after four cycles CFU-S recovered to only 10% of normal. BB-10010 afforded little benefit in the first treatment cycle, but by the end of the fourth cycle CFU-S still recovered to 35% of normal. MRA was reduced to 7% of normal by the irradiation protocol-about half that maintained by BB-10010 protection. We conclude that BB-10010 (MIP-1 alpha) reduces the degree of accumulated haemopoietic stem cell damage following repeated non-cell cycle-specific cytotoxic insults-a principle which should be valuable in repeated clinical cytotoxic therapy regimens.

The Third Human Homolog of a Murine Gene Encoding an Inhibitor of Stem Cell Proliferation Is Truncated and Linked to a CpG Island-Containing Upstream Sequence

The murine gene, MIP1 alpha, encodes a cytokine (macrophage inflammatory protein 1 alpha) that inhibits the proliferation of bone marrow stem cells. Two human homologs have been characterized, G0S19-1 and G0S19-2. Like MIP1 alpha, these genes contain three exons, the first of which encodes a hydrophobic signal sequence. The existence of a third human G0S19 gene, present in one in four individuals, has been predicted from restriction enzyme analyses. This paper reports that a previously identified human genomic clone containing a G0S19 sequence (G0S19-3), corresponds to the third gene. However, the first G0S19 exon is missing. The sequence differs from those of G0S19-1 and G0S19-2 upstream of a point 31 nucleotides from the junction of the first intron with the second exon. This upstream sequence contains a CpG island and is named "CpG island-containing upstream sequence," CUS. Apart from the G0S19-3-associated copy found only in individuals with the third G0S19 gene, all individuals have one DNA species hybridizing strongly to a CUS-specific probe and at least two less strongly hybridizing species. The CUS has potential binding sites for transcription factors AP-1, AP-2, AP-3, AP-4, and Sp1, a Donehower conserved repetitive element, and motifs characteristic of cytokine, oncogene, and retroviral promoters. Thus, the CUS might promote the transcription of sequences with which it became associated. We suggest that the CUS-G0S19-3 sequence was generated by recombination between a G0S19-2 gene and a member of a novel CUS-associated gene family.

A Set of Human Putative Lymphocyte G0/G1Switch Genes Includes Genes Homologous to Rodent Cytokine and Zinc Finger Protein-Encoding Genes

Lymphocyte G0/G1 switch genes (G0S genes) are potential oncogenes and may regulate, be regulated by, or be coordinately regulated with, latent lymphotropic viruses. To identify these genes, a cDNA library was prepared from blood mononuclear cells that had been cultured for 2 hr with a T-cell mitogen (lectin) and cycloheximide. Eight differentially hybridizing recombinants were characterized by RNA and DNA blotting and sequencing. One cDNA (G0S7) corresponded to the oncogene c-fos. Another cDNA (G0S19) was homologous (70%) to a cDNA encoding a murine inhibitor of stem cell proliferation (the cytokine MIP1 alpha) and, less closely, to other members of the "small inducible" secreted protein-encoding gene family. Whereas cDNA hybridization to genomic DNA blots indicated a small subfamily of G0S19 genes, simple patterns of bands indicated that most cDNAs, including G0S30 cDNA, corresponded to single-copy genes. The 3' noncoding sequence of G0S30 cDNA was homologous (87-89%) to the 3' noncoding sequences of certain rodent genes (NGFI-A, Krox24, EGR1) that encode zinc finger proteins (putative transcriptional regulators). This degree of evolutionary conservation suggests an important function for the 3' noncoding region. The 3' noncoding regions of some cDNAs contained the TTATTTAT (mRNA destabilization) element. The corresponding RNAs each formed doublets in agarose gels. Previous studies of c-fos RNA from HeLa cells indicate that this is due to cycloheximide-dependent stabilization of poly(A) tails. Our results reveal the power of cycloheximide enrichment in isolating what would appear to be significant low-abundance mRNAs.

Three Human Homologs of a Murine Gene Encoding an Inhibitor of Stem Cell Proliferation

The G0S19 genes are members of the "small inducible" family of genes, which have similar exon-intron organizations and encode secreted proteins with similar dispositions of cysteine and proline residues. G0S19-1 mRNA is increased shortly after the addition of lectin or cycloheximide to cultured human blood mononuclear cells. The cDNA sequence is homologous to that of a murine gene encoding an inhibitory cytokine (MIP1 alpha/SCI), which decreases hemopoietic stem cell proliferation. The homology extends to the 3' noncoding region, which contains two conserved elements: (i) GGGACTCTTC, a potential transcription factor NF chi B-binding site, and (ii) TTTTGTAATTTATTTT, which is found in some related genes (e.g., that encoding the immediate early protein ornithine decarboxylase). A similar but complementary sequence is present in human immunodeficiency virus. Two of the three human genes that hybridize to G0S19-1 cDNA were sequenced. G0S19-1 has 5' AP1-like recognition elements as found in some other phorbol ester-responsive genes (e.g., c-fos). G0S19-2 has a 5' Alu sequence, but is likely to be expressed because of the conservation of sections of the gene believed to be important for function. The 5' flanks of both genes contain the nucleotide motifs CK-2 and SRE, indicating cytokine-like genes with the potential to respond to growth factors. G0S19-1 is the main G0S19 gene expressed in adult T lymphocytes and may encode a homeostatic negative regulator of the size of cell populations (or subpopulations) which are derived ultimately from marrow stem cells. As such, it is a potential antioncogene.

An Inhibitor of Stem Cell Proliferation in Normal Bone Marrow

A saline extract from normal bone marrow cells having a molecular weight in the range 50000-1000000 daltons has been found to protect rapidly proliferating haemopoietic spleen colony forming cells (CFUs) from the lethal effects of large doses of tritiated thymidine. This extract is non-toxic to the cells. It is not found in regenerating marrow where the CFUs population is rapidly proliferating. Its effect appears to be specific for the CFUs since it has no effect on the proliferation of its close descendant, the granulocytic precursor cell (CFUc), and no effect on the average cytoplasmic structuredness of the whole bone marrow cell population. The active material is probably protein since it is degraded by trypsin.