Bernard A. Roos

Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential

We report here the isolation of a population of non-transformed pluripotent human cells from bone marrow after a unique expansion/selection procedure. This procedure was designed to provide conditions resembling the in vivo microenvironment that is home for the most-primitive stem cells. Marrow-adherent and -nonadherent cells were co-cultured on fibronectin, at low oxygen tension, for 14 days. Colonies of small adherent cells were isolated and further expanded on fibronectin at low density, low oxygen tension with 2% fetal bovine serum. They expressed high levels of CD29, CD63, CD81, CD122, CD164, hepatocyte growth factor receptor (cMet), bone morphogenetic protein receptor 1B (BMPR1B), and neurotrophic tyrosine kinase receptor 3 (NTRK3) and were negative for CD34, CD36, CD45, CD117 (cKit) and HLADR. The embryonic stem cell markers Oct-4 and Rex-1, and telomerase were expressed in all cultures examined. Cell-doubling time was 36 to 72 hours, and cells have been expanded in culture for more than 50 population doublings. This population of cells was consistently isolated from men and women of ages ranging from 3- to 72-years old. Colonies of cells expressed numerous markers found among embryonic stem cells as well as mesodermal-, endodermal- and ectodermal-derived lineages. They have been differentiated to bone-forming osteoblasts, cartilage-forming chondrocytes, fat-forming adipocytes and neural cells and to attachment-independent spherical clusters expressing genes associated with pancreatic islets. Based on their unique characteristics and properties, we refer to them as human marrow-isolated adult multilineage inducible cells, or MIAMI cells. MIAMI cells proliferate extensively without evidence of senescence or loss of differentiation potential and thus may represent an ideal candidate for cellular therapies of inherited or degenerative diseases.

Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow

Mesenchymal stem cells (MSCs) residing in bone marrow (BM) are the progenitors for osteoblasts and for several other cell types. In humans, the age‐related decrease in bone mass could reflect decreased osteoblasts secondary to an age‐related loss of osteoprogenitors. To test this hypothesis, BM cells were isolated from vertebral bodies of thoracic and lumbar spine (T1–L5) from 41 donors (16 women and 25 men) of various ages (3–70 years old) after death from traumatic injury. Primary cultures were grown in alpha modified essential medium with fetal bovine serum for 13 days until adherent cells formed colonies (CFU‐Fs). Colonies that stained positive for alkaline phosphatase activity (CFU‐F/ALP+) were considered to have osteogenic potential. BM nucleated cells were plated (0.5, 1, 2.5, 5, or 10 × 106 cells/10‐cm dish) and grown in dexamethasone (Dex), which promotes osteoblastic differentiation. The optimal plating efficiency using BM‐derived cells from donors of various ages was 5 × 106 cells/10‐cm dish. BM‐derived cells were also grown in the absence of Dex at this plating density. At the optimal plating density, in the presence of Dex, the number of CFU‐F/ALP+ present in the BM of the younger donors (3–36 years old) was 66.2 ± 9.6 per 106 cells (mean ± SEM), but only 14.7 ± 2.6 per 106 cells in the older donors (41–70 years old). With longer‐term culture (4–5 weeks) of these BM cells in medium containing 10 mM β‐glycerophosphate and 100 μg/ml ascorbic acid, the extracellular matrix mineralized, a result consistent with mature osteoblastic function. These results demonstrate that the number of MSCs with osteogenic potential (CFU‐F/ALP+) decreases early during aging in humans and may be responsible for the age‐related reduction in osteoblast number. Our results are particularly important in that the vertebrae are a site of high turnover osteoporosis and, possibly, the earliest site of bone loss in age‐related osteoporosis.

Interventions to Reduce Hospitalizations from Nursing Homes: Evaluation of the INTERACT II Collaborative Quality Improvement Project

A substantial proportion of hospitalizations of nursing home (NH) residents may be avoidable. Medicare payment reforms, such as bundled payments for episodes of care and value‐based purchasing, will change incentives that favor hospitalization but could result in care quality problems if NHs lack the resources and training to identify and manage acute conditions proactively. Interventions to Reduce Acute Care Transfers (INTERACT) II is a quality improvement intervention that includes a set of tools and strategies designed to assist NH staff in early identification, assessment, communication, and documentation about changes in resident status. INTERACT II was evaluated in 25 NHs in three states in a 6‐month quality improvement initiative that provided tools, on‐site education, and teleconferences every 2 weeks facilitated by an experienced nurse practitioner. There was a 17% reduction in self‐reported hospital admissions in these 25 NHs from the same 6‐month period in the previous year. The group of 17 NHs rated as engaged in the initiative had a 24% reduction, compared with 6% in the group of eight NHs rated as not engaged and 3% in a comparison group of 11 NHs. The average cost of the 6‐month implementation was

Gap-junctional communication is required for the maturation process of osteoblastic cells in culture.

7,700 per NH. The projected savings to Medicare in a 100‐bed NH were approximately

Anabolic or Catabolic Responses of MC3T3-E1 Osteoblastic Cells to Parathyroid Hormone Depend on Time and Duration of Treatment

125,000 per year. Despite challenges in implementation and caveats about the accuracy of self‐reported hospitalization rates and the characteristics of the participating NHs, the trends in these results suggest that INTERACT II should be further evaluated in randomized controlled trials to determine its effect on avoidable hospitalizations and their related morbidity and cost.

Inhibition of Gap-Junctional Communication Induces the Trans-differentiation of Osteoblasts to an Adipocytic Phenotype in Vitro

Osteoblastic cells in long-term culture undergo a phenotypic maturation process leading to extracellular matrix (ECM) production and bone nodule (BN) formation. Cell-to-cell communication via gap junctions (GJC) can be detected between osteoblastic cells within 24 h of plating. We evaluated, in long-term cultures of osteoblastic cells, the effect of inhibiting GJC on the phenotypic maturation process and the expression of specific genes associated with this process. MC3T3-E1 cells were plated, and, after 24 h (day 0), cells were exposed to 18-alpha-glycyrrhetinic acid (AGA), a nontoxic reversible inhibitor of GJC. GJC, alkaline phosphatase (AP) activity, BN formation, and the relative level of transcripts encoding osteocalcin (OC), bone sialoprotein (bSP), osteopontin (OP), collagen alpha1 type I (alpha1ICol), and elongation factor-1a (EF1a) were evaluated on day 0 and every 4-7 days thereafter through day 30. GJC was assessed by fluorescent dye transfer. Gene expression was analyzed by northern blot and semiquantitative reverse transcription-polymerase chain reaction. GJC was detectable at day 0 and increased with time in culture. AGA (100 micromol/L) strongly inhibited GJC at all timepoints tested. Moreover, AGA-exposed cells showed a dose-dependent decrease in AP activity and a delay in the appearance of BN. This delayed phenotypic expression coincided with an inhibitory effect on the expression of the osteoblast-specific genes OC and bSP. Expression of alpha1ICol mRNA was also affected, but to a lesser extent, whereas OP and EF1a were not affected. Similar results were obtained with oleamide, an additional reversible inhibitor of GJC. In contrast, cells exposed to either vehicle or 100 micromol/L glycyrrhizic acid (a noninhibitory glycoside of 18-beta-glycyrrhetinic acid) were indistinguishable from untreated cells for all parameters evaluated. We conclude that GJC inhibition interferes with the maturation process of osteoblastic cells in culture, possibly by affecting signals regulating the expression of genes involved in the maturation/differentiation of the osteoblastic phenotype.

A role for GATA transcription factors in the androgen regulation of the prostate-specific antigen gene enhancer

We have investigated signaling (cAMP) and anabolic responses (mineralization of extracellular matrix [ECM]) to parathyroid hormone (PTH) in long‐term (30 days) cultures of MC3T3‐E1 cells, a murine model of osteoblast differentiation. Expression of PTH/PTH–related peptide receptor (PTH1R) mRNA is detected early and remains relatively constant for 2 weeks with somewhat higher levels observed during the second half of the culture period. In contrast to the relatively stable PTH1R mRNA expression, the cAMP response to PTH varies markedly with no response at day 5 and a marked response (80‐fold versus control) by day 10. Responsiveness to PTH remains elevated with fluctuations of 30‐ to 80‐fold stimulation throughout the remainder of the culture period. The timing and duration of PTH treatment to achieve in vitro mineralization of ECM was evaluated. When continuous PTH treatment was initiated before day 20, mineralization decreased. If continuous PTH treatment began on or after day 20, mineralization was unaffected. However, if treatment began on day 20 and then stopped on day 25, mineralization on day 30 was increased 5‐fold. This mineralization response to intermittent PTH was confirmed in primary cultures of murine and human osteoblastic cells. These data provide a potential basis for understanding the differential responses to PTH (anabolic versus catabolic) and indicate the developmental temporal variance of anabolic and catabolic responses. Since cAMP signaling was relatively unchanged during this interval (day 10–30) and stimulation of adenylate cyclase only partially mimicked the PTH effect on increased mineralization, other signaling pathways are likely to be involved in order to determine the specific anabolic response to short‐term PTH treatment during the differentiation process.

Neuroendocrine peptides in the prostate

Osteoblasts and adipocytes are thought to differentiate from a common stromal progenitor cell. These two phenotypically mature cell types show a high degree of plasticity, which can be observed when cells are grown under specific culture conditions. Gap junctions are abundant among osteoblastic cellsin vivo and in vitro, whereas they are down-regulated during adipogenesis. Gap junctional communication (GJC) modulates the expression of genes associated with the mature osteoblastic phenotype. Inhibition of GJC utilizing 18-α-glycyrrhetinic acid (AGRA) blocks the maturation of pre-osteoblastic cells in vitro. Moreover, cytoplasmic lipid droplets are detectable at the end of the culture period, suggesting that GJC inhibition may favor an adipocytic phenotype. We used several human osteoblastic cell lines, as well as bone-derived primary osteoblastic cells, to show that confluent cultures of human osteoblastic cells grown under osteogenic conditions developed an adipocytic phenotype after 3 days of complete inhibition of GJC using AGRA or oleamide, two dissimilar nontoxic reversible inhibitors. Development of an adipogenic phenotype was confirmed by the accumulation of triglyceride droplets and the increase in mRNA expression of the adipocytic markers peroxisome proliferator-activated receptor γ2 and lipoprotein lipase. Glycyrrhizic acid, a noninhibitory AGRA analog, or α-bromopalmitate, a nondegradable fatty acid, had no effect. Modulation of skeletal GJC may represent a new pharmacological target by which inhibition of marrow adipogenesis can take place with the parallel enhancement of osteoblastogenesis, thus providing a novel therapeutic approach to the treatment of human age-related osteopenic diseases and postmenopausal osteoporosis.

Optimal Frequency, Displacement, Duration, and Recovery Patterns to Maximize Power Output Following Acute Whole-Body Vibration

The transgenic mouse line Ggamma/T-15 containing the fetal globin promoter linked to SV40 T antigen unexpectedly results in androgen-independent prostate carcinomas. Given the key role of GATA-1 transcription factor in fetal globin gene promoter activity, we investigated whether specific GATA family members are expressed in the prostate and whether they can regulate prostate-specific genes. We found that GATA-2 and -3 are the predominant GATA family members expressed in human and mouse prostate and that GATA mRNA levels are not regulated by androgen. We identified six GATA sites flanking an androgen-response element located in the far-upstream enhancer of the prostate-specific antigen (PSA) gene. These GATA sites are targets for GATA factors and are essential for optimal androgen induction of transfected PSA enhancer/promoter plasmids in LNCaP, a PSA and androgen receptor expressing human prostate cancer cell line. Our results suggest that prostatic GATA-2 and -3 are involved in the androgen regulation of the PSA gene.

Gap-junctional communication mediates parathyroid hormone stimulation of mineralization in osteoblastic cultures.

Circulating androgens are required for normal growth and maintenance of function of the prostate. However, the prostate also contains neuroendocrine peptides, found either in nerve terminals or in prostatic neuroendocrine cells, which are likely to regulate prostate growth or function. The neuronal peptides are likely to participate in the regulation of the synthesis and secretion of prostatic secretory products. While the function of the neuroendocrine cells is undefined, there is evidence for growth-regulating effects of several neuroendocrine cell peptides. Since neuroendocrine differentiation has been correlated with tumor grade and poor prognosis in prostate cancer, the peptide products of the neuroendocrine cells may influence cancer cell replication as well. Recent evidence in other tissues suggests that peptide hormone receptor second-messenger systems may interact with steroid receptors to modulate their actions. These findings raise the possibility that prostatic neuroendocrine peptides may modulate the response of prostate to androgens.