Combination of hTERT and bmi-1, E6, or E7 Induces Prolongation of the Life Span of Bone Marrow Stromal Cells from an Elderly Donor without Affecting Their Neurogenic Potential

Abstract

Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro.Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.Murine and human bone marrow stromal cells differentiate into osteoblasts (2), chondrocytes (13), skeletal myocytes, adipocytes, and cardiomyocytes (24) in vitro and thus are a useful cell source for bone regeneration (26) and in vivo cardiovasculogenesis (11). However, recent studies suggest that bone marrow stromal cells can also differentiate into a neuronal lineage (22), and murine bone marrow-derived multipotent adult progenitor cells differentiate into dopaminergic neuronal cells (16). Since the use of bone marrow stromal cells entails no ethical or immunological problems, and bone marrow aspiration is an established routine procedure, they may be a useful source of cells for transplantation.Large numbers of cells may be necessary for repairing damaged human tissues to restore function. However, there have been no reports of a sufficient number of differentiated neurons ever having been obtained from human marrow stromal cells. One reason is that normal human cells undergo a limited number of divisions in culture and then enter a nondividing state referred to as "senescence." Senescence is classified into two categories: "stress-induced premature senescence," or "telomere-independent senescence," and "replicative senescence," or "telomere-dependent senescence" (3, 5, 38). p16 Ink4a (p16), a cyclin-dependent kinase (CDK) inhibitor, is induced by certain oncogenes and other damage or stress signals and is required for "premature senescence" in human mammary epithelial cells and keratinocytes. p16 inhibits dephosphorylation of pRb by Cdk4/6-cyclin D, and hypophosphorylated pRb actively represses the genes required for the S phase by sequestering the E2F transcription factors. "Replicative senescence" is caused by telomere size reduction during successive cell divisions because of the chromosome...

Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation

“…Cell life span of menstrual blood cells is relatively short when compared with human fetal cells (Imai et al, 1994;Terai et al, 2005), and this shorter cell life span may be attributed to shorter telomere length of adult cells (i.e., endometrial stromal cells) at the start of cell cultivation, as is the case with hematopoietic stem cells (Suda et al, 1984). Menstrual blood-derived cells had a high replicative ability similar to progenitors or stem cells that display a longterm self-renewal capacity and had a much higher growth rate in our experimental conditions than marrow-derived stromal cells (Mori et al, 2005). In addition, the myogenic potential of menstrual blood-derived cells, i.e., a high frequency of desmin-positive cells after induction, is much greater than expected.…”

“…Immunohistochemical analysis was performed as previously described (Mori et al, 2005). Briefly, the sections were incubated for 1 h at room temperature with mouse mAb against vimentin (Cone V9, DakoCytomation, Fort Collins, CO).…”

“…Western blot analysis was performed as previously described (Mori et al, 2005). Blots were incubated with primary antibodies (desmin, myogenin [Clone F5D,Santa Cruz Biotechnology], and dystrophin [NCL-DYSA, Novocastra]) for 1-2 h at room temperature.…”

“…Immunocytochemical analysis was performed as previously described (Mori et al, 2005), with antibodies to skeletal myosin (Sigma, St. Louis, MO; product no. M 4276), MF20 (which reacts with all sarcomere myosin in striated muscles, Developmental Studies Hybridoma Bank, University of Iowa, IA), ␣-sarcomeric actin (Sigma, product no.…”

SOX10 is a novel marker of acinus and intercalated duct differentiation in salivary gland tumors: a clue to the histogenesis for tumor diagnosis

“…Images were collected with a confocal microscope system (LSM 510 META; Zeiss). 9,10 SOX10-Venus BAC Transgenic Mice SOX10-Venus bacterial artificial chromosome (BAC) transgenic mice have been described previously. 11…”