Heterogeneity within the hematopoietic stem cell compartment: evidence for a marrow-seeding stem cell distinct from CFU-s

Using a chromosome marker within a syngeneic system, we investigated the seeding characteristics of murine hematopoietic stem cells after transplantation to irradiated hosts. The chromosome-marked test cells were allowed to compete with normal marrow cells in repopulating the spleen and marrow of irradiated mice. Although the seeding behavior of normal marrow could be predicted from the number of colony-forming units-spleen (CFU-s) transplanted, the marrow seeding of melphalan-treated marrow was 7-fold greater than expected. Repopulation of marrow by spleen cells was less effective than expected from the CFU-s content, while the reverse was true after repopulation by fetal liver cells. These differences were emphasized after treatment of cell donors with melphalan. The results were due primarily to differences in the lodging properties of the transplanted cells, those seeding in the marrow were less sensitive to melphalan than CFU-s. In some instances marrow-repopulating ability could be separated from peak CFU-s activity on a density gradient, suggesting a marrow-repopulating cell exists that is distinct from CFU-s.     

A new puzzling role of p53 in mouse embryonic stem cells

Comment on: Lee KH, et al. Proc Natl Acad Sci USA 2010; 107:69-74.     

Liver stem cells: a two compartment system

Hepatocytes and biliary epithelia are phenotypically very dissimilar, but share a common ancestry. Hepatocytes regenerate very efficiently, and their division potential indicates that many of them are functional stem cells. When hepatocyte-damaging agents also impair the regenerative ability of surviving hepatocytes, a potential stem cell system of biliary origin is activated to generate new hepatocytes — a reversal of ontogeny. Now both bile duct derived cells and hepatocytes can be isolated from the liver, genetically modified in vitro and returned to their in vivo origins where, after considerable population expansion, they can function as hepatocytes — paving the way for ex vivo gene therapy.     

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.     

p53: The barrier to cancer stem cell formation

The role of p53 as the “guardian of the genome” in differentiated somatic cells, triggering various biological processes, is well established. Recent studies in the stem cell field have highlighted a profound role of p53 in stem cell biology as well. These studies, combined with basic data obtained 20 years ago, provide insight into how p53 governs the quantity and quality of various stem cells, ensuring a sufficient repertoire of normal stem cells to enable proper development, tissue regeneration and a cancer free life. In this review we address the role of p53 in genomically stable embryonic stem cells, a unique predisposed cancer stem cell model and adult stem cells, its role in the generation of induced pluripotent stem cells, as well as its role as the barrier to cancer stem cell formation.     

p53 and cancer stem cells: The mevalonate connexion

Comment on: Freed-Pastor WA, et al. Cell 2012; 148:244-58 and Ginestier C, et al. Stem Cells 2012; 1327-37.     

Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation

Connexin 43 knockout (Cx43alpha1KO) mice exhibit germ cell deficiency, but the underlying cause for the germ cell defect was unknown. Using an Oct4-GFP reporter transgene, we tracked the distribution and migration of primordial germ cells (PGCs) in the Cx43alpha1KO mouse embryo. Analysis with dye injections showed PGCs are gap-junction-communication competent, with dye coupling being markedly reduced in Cx43alpha1-deficient PGCs. Time-lapse videomicroscopy and motion analysis showed that the directionality and speed of cell motility were reduced in the Cx43alpha1KO PGCs. This was observed both in E8.5 and E11.5 embryos. By contrast, PGC abundance did not differ between wild-type and heterozygous/homozygous Cx43alpha1KO embryos until E11.5, when a marked reduction in PGC abundance was detected in the homozygous Cx43alpha1KO embryos. This was accompanied by increased PGC apoptosis and increased expression of activated p53. Injection of alpha-pifithrin, a p53 antagonist, inhibited PGC apoptosis and prevented the loss of PGC. Analysis using a cell adhesion assay indicated a reduction in beta1-integrin function in the Cx43alpha1KO PGCs. Together with the abnormal activation of p53, these findings suggest the possibility of anoikis-mediated apoptosis. Overall, these findings show Cx43alpha1 is essential for PGC survival, with abnormal p53 activation playing a crucial role in the apoptotic loss of PGCs in the Cx43alpha1KO mouse embryos.     

Same stem cells but different: Isogenic embryonic stem cells harboring different p53 mutants

Comment on: Wei QX, et al. Cell Cycle 2011; 10:1261-70.     

Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.     

Pluripotent stem cells from the adult mouse inner ear

In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.