No abstract

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.

Aging of the human skeleton is characterized by decreased bone formation and bone mass and these changes are more pronounced in patients with osteoporosis. As osteoblasts and adipocytes share a common precursor cell in the bone marrow, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis is the result of enhanced adipognesis versus osteoblastogenesis from precursor cells in the bone marrow. Thus, we examined iliac crest bone biopsies obtained from 53 healthy normal individuals (age 30-100) and 26 patients with osteoporosis (age 52-92). Adipose tissue volume fraction (AV), hematopoietic tissue volume fraction (HV) and trabecular bone volume fraction (BV) were quantitated as a percentage of total tissue volume fraction (TV) (calculated as BV + AV + HV) using the point-counting method. We found an age-related increase in AV/TV (r = 0.53, P < 0.001, n = 53) and an age-related decline in BV/TV (r = -0.46, P < 0.001, n = 53) as well as in the HV/TV (r -0.318, P < 0.05, n = 53). There was an age-related inverse correlation between BV/TV and AV/TV (r = -0.58, P < 0.001). No significant correlation between the AV/TV and the body mass index (r = 0.06, n.s., n = 52) was detectable. Compared with age-matched controls, patients with osteoporosis exhibited an increased AV/TV (P < 0.05) and decreased BV/TV (P < 0.05) but no statistically significant difference in HV/TV. Our data support the hypothesis that with aging and in osteoporosis an enhanced adipogenesis is observed in the bone marrow and that these changes are inversely correlated to decreased trabecular bone volume. The cellular and molecular mechanisms mediating these changes remain to be determined.

Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the result of enhanced adipogenesis and decreased osteoblastogenesis from the MSCs. Thus, cultures of MSCs were established from young donors (age 18-42, n = 34), elderly healthy donors (age 66-78, n = 20), and patients with OP (age 58-76, n = 15). Cells were cultured for 2 weeks in an adipogenic medium (containing 15% horse serum and 100 nM dexamethasone), osteogenic medium (containing 10% fetal calf serum [FCS] and 10 nM calcitriol), or control medium (10% FCS). The MSCs were identified by their abilities to form colonies. Total number of colonies, osteoblastic colonies stained positive for alkaline phosphatase (AP+), and adipocytic colonies containing adipocytes (Ad+) were quantitated. In addition, steady state mRNA levels of gene markers of adipocytic and osteoblastic phenotypes were determined using reverse-transcriptase polymerase chain reaction (RT-PCR). The adipogenic and osteogenic media induced cell differentiation and the expression of adipocytic and osteoblastic lineage-specific markers, respectively. We found no age-related changes in the osteoblastic or adipocytic colony formation or the steady state levels of mRNA of the adipogenic or osteogenic gene markers. Cells obtained from patients with OP showed a pattern of differentiation similar to those of age-matched controls. In conclusion, MSCs maintain their differentiation potential during aging and in patients with OP. Other mechanisms responsible for age-related decrease in bone formation need to be determined.

No abstract

Our results suggest that the absence or reduced volume of the sebaceous gland may play a role in the pathogenesis of HS. The presence of fibrosis suggests that sebaceous glands are obliterated early in the pathogenesis of HS.

Tumor necrosis factor-alpha (TNF-alpha) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-alpha-encoding mRNA, however, is not increased in psoriatic skin, and it remains unclear whether intervention strategies based on RNA interference (RNAi) are therapeutically relevant. To test this hypothesis the present study describes first the in vitro functional screening of a panel of short hairpin RNAs (shRNAs) targeting human TNF-alpha mRNA and, next, the transfer of the most potent TNF-alpha shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-alpha shRNA treatment leads to amelioration of the psoriasis phentotype in the model, as documented by reduced epidermal thickness, normalization of the skin morphology, and reduced levels of TNF-alpha mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-alpha shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-alpha shRNAs in human skin. These findings validate TNF-alpha mRNA as a target molecule for a potential persistent RNA-based treatment of psoriasis and establish the use of small RNA effectors as a novel platform for target validation in psoriasis and other skin disorders.

Interconversion of bone marrow osteoblasts and adipocytes has been reported previously. However, the osteogenic potential of extramedullary adipocytes is not known. Thus, we incubated a pure culture of human subcutaneous adipocytes in control medium for 1-2 weeks. Afterward, the cells were incubated in either osteoblast medium (OB medium) containing various combinations of calcitriol, dexamethasone, ascorbic acid, and beta-glycerophosphate or in adipocyte medium (AD medium) containing HEPES, biotin, pantothenate, insulin, triiodothyronine, dexamethasone, and isobutylmethylxanthine for 4 weeks. Expression of osteoblastic and adipocytic phenotypes was examined by determination of lineage-specific mRNA markers and in vitro adipocyte and osteoblast formation. Cells were also implanted, mixed with hydroxyapatite-tricalcium phosphate powder, in the subcutaneous tissue of immunodeficient mice in order to assess in vivo bone formation potential. One week after incubation in control medium, cells formed fusiform elongated fibroblast-like cells. In OB medium, cells stained positive for alkaline phosphatase (AP) and expressed mRNAs encoding Cbfa1/Runx2, AP, and osteocalcin. In AD medium cells reacquired adipocyte morphology with multilocular lipid-filled cells. Also, the cells expressed adipocyte-specific mRNA markers: lipoprotein lipase and peroxisome proliferator-activated receptor gamma2. Bone was formed only in the in vivo implants of cells incubated in OB medium. In conclusion, extramedullary adipocytes can transdifferentiate to bone-forming cells. Because of their ease of isolation, adipocytes may be good candidates for tissue-engineering protocols aimed at creating bone tissue for the repair of nonunion fractures and large bone defects.