The osmotic characteristics of human fetal liver-derived hematopoietic stem cell candidates

Hematopoietic stem cells derived from fetal liver have promising therapeutic potential for allotransplantation but require a specific protocol to minimize the damage produced by cryopreservation procedures. In this study, a fundamental approach was applied for designing a cell preservation protocol. To this end, the biophysical characteristics that describe the osmotic reaction of CD34(+)CD38(-) human fetal liver stem cell candidates were studied using fluorescent microscopy. The osmotically inactive volume of the stem cell candidates was determined as 48% of the isotonic volume. The permeability coefficients for water and Me(2)SO were determined at T = +22 degree C: L(p) = 0.27 +/- 0.03 microm x min(-1)atm(-1), P(Me(2)SO)) = 2.09 +/- 0.85 x 10 (-4) cm x min(-1), sigma (Me(2)SO)) = 0.63 +/- 0.03 and at T = +12 degree C: L(p) = 0.15 +/-0.02 microm x min(-1)atm(-1), P(Me(2)SO)) = 6.44 +/-1.42 x 10 (-5) cm x min(-1), sigma (Me(2)SO)) = 0.46 +/- 0.05. The results obtained suggest that post-hypertonic and hypotonic stress are the possible reasons for damage to a CD34(+)CD38(-) cell during the cryopreservation procedure.     

Long-term molecular and cellular stability of human neural stem cell lines

Human Neural Stem Cells (hNSCs) are excellent candidates for in vitro and in vivo molecular, cellular, and developmental research, and also for ex-vivo gene transfer and cell therapy in the nervous system. However, hNSCs are mortal somatic cells, and thus invariably enter an irreversible growth arrest after a finite number of cell divisions in culture. It has been proposed that this is due to telomere shortening. Here, we show that long-term cultured (up to 4 years) v-myc perpetuated hNSC lines do preserve short but stable and homogeneous telomeres (TRF and Q-FISH determinations). hNSC lines (but not strains) express high levels of telomerase activity, which is activated by v-myc, as demonstrated here. Telomerase activity is not constitutive, becoming non-detectable after differentiation (in parallel to v-myc down-regulation). hNSC lines also maintain a stable cell cycle length, mitotic potential, differentiation and neuron generation capacity, and do not express senescence-associated beta-galactosidase over years, as studied here. These data, collectively, help to explain the immortal nature of v-myc-perpetuated hNSC lines, and to establish them as excellent research tools for basic and applied neurobiological and translational studies.     

Signals from damaged but not undamaged skeletal muscle induce myogenic differentiation of rat bone-marrow-derived mesenchymal stem cells

The regenerative capacity of skeletal muscle has been usually attributed to resident satellite cells, which, upon activation by local or distant stimuli, initiate a myogenic differentiation program. Although recent studies have revealed that bone-marrow-derived progenitor cells may also participate in regenerative myogenesis, the signals and mechanisms involved in this process have not been elucidated. This study was designed to investigate whether signals from injured rat skeletal muscle were competent to induce a program of myogenic differentiation in expanded cultures of rat bone-marrow-derived mesenchymal stem cells (MSC). We observed that the incubation of MSC with a conditioned medium prepared from chemically damaged but not undamaged muscle resulted in a time-dependent change from fibroblast-like into elongated multinucleated cells, a transient increase in the number of MyoD positive cells, and the subsequent onset of myogenin, alpha-actinin, and myosin heavy chain expression. These results show that damaged rat skeletal muscle is endowed with the capacity to induce myogenic differentiation of bone-marrow-derived mesenchymal progenitors.     

Protein structure preference, tRNA copy number, and mRNA stem/loop content

From statistical analyses of protein sequences for humans and Escherichia coli we found that the messenger RNA segment of m-codons (for m=2 to 6) with average high tRNA copy number (TCN) (larger than approximately 10.5 for humans or approximately 1.95 for E. coli) preferably code for the alpha helix and that with low TCN (smaller than approximately 7.5 for humans or approximately 1.7 for E. coli) preferably code for coil. Between them there is an intermediate region without correlation to structure preference. For the beta strand the preference/ avoidance tendency is not obvious. All strong preference-modes of TCN for protein secondary structures have been deduced. The mutual interaction between two factors--protein secondary structural type and codon TCN--is tested by F distribution. A phenomenological model on the relation between structure preference and translational efficiency or accuracy is proposed. It is pointed out that the structure preference of codons is related to the distribution of mRNA stem/loop content in three TCN regions.     

Mesenchymal stem cells from cryopreserved human umbilical cord blood

Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages, but the presence of mesenchymal stem cells (MSCs) in UCB has been disputed and it remains to be validated. In this study, we examined the ability of cryopreserved UCB harvests to produce cells with characteristics of MSCs. We were able to obtain homogeneous plastic adherent cells from the mononuclear cell fractions of cryopreserved UCB using our culture conditions. These adherent cell populations exhibited fibroblast-like morphology and typical mesenchymal-like immunophenotypes (CD73+, CD105+, and CD166+, etc.). These cells presented the self-renewal capacity and the mesenchymal cell-lineage potential to form bone, fat, and cartilage. Moreover, they expressed mRNAs of multi-lineage genes including SDF-1, NeuroD, and VEGF-R1, suggesting that the obtained cells had the multi-differentiation capacity as bone marrow-derived MSCs. These results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and therapeutic applications.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Chondrogenic differentiation of human mesenchymal stem cells cultured in a cobweb-like biodegradable scaffold

Human mesenchymal stem cells (MSCs) were cultured in vitro in a cobweb-like biodegradable polymer scaffold: a poly(dl-lactic-co-glycolic acid)-collagen hybrid mesh in serum-free DMEM containing TGF-beta3 for 1-10 weeks. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The ability of the cells to express gene encoding type I collagen decreased, whereas its ability to express type II collagen and aggrecan increased. Histological examination by HE staining indicated that the cells showed fibroblast morphology at the early stage and became round after culture for 4 weeks. The cartilaginous matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. In addition, a homogeneous distribution of cartilaginous extracellular matrices was detected around the cells. These results suggest the chondrogenic differentiation of the mesenchymal stem cells in the hybrid mesh. The PLGA-collagen hybrid mesh enabled the aggregation of mesenchymal stem cells and provided a promotive microenvironment for the chondrogenic differentiation of the MSCs.     

A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix

Mesenchymal stem cells (MSC) show a very short proliferative life span and readily lose the differentiation potential in culture. However, the growth rate and the proliferative life span of the stem cells markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix, which was produced by PYS-2 cells or primary endothelial cells. Furthermore, the stem cells expanded on the extracellular matrix, but not those on plastic tissue culture dishes, retained the osteogenic, chondrogenic, and adipogenic potential throughout many mitotic divisions. The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor. Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies, which require a large number of stem or progenitor cells.     

Adipocyte differentiation of multipotent cells established from human adipose tissue

In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.e., expression of specific molecular markers, lipolytic response to agonists of beta-adrenoreceptors (beta2-AR agonist > beta1-AR agonist > beta3-AR agonist) and to the atrial natriuretic peptide, insulin-stimulated glucose transport, and secretion of leptin and adiponectin. hMADS cells were able to respond to drugs as inhibition of adipocyte differentiation was observed in the presence of prostaglandin F2alpha, tumour necrosis factor-alpha, and nordihydroguaiaretic acid, a natural polyhydroxyphenolic antioxidant. Thus, for the first time, human adipose cells with normal karyotype and indefinite life span have been established. They represent a novel and valuable tool for studies of fat tissue development and metabolism.