Low Oxygen Modulates Multiple Signaling Pathways, Increasing Self-Renewal, While Decreasing Differentiation, Senescence, and Apoptosis in Stromal MIAMI Cells.

Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and ß-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells.

Hepatocyte growth factor and alternative splice variants - expression, regulation and implications in osteogenesis and bone health and repair.

INTRODUCTION: Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. These pluripotent cells secrete hepatocyte growth factor (HGF), which regulates cell growth, survival, motility, migration, mitogenesis and is important for tissue development/regeneration. HGF has four splice variants, NK1, NK2, NK3, and NK4 which have varying functions and affinities for the HGF receptor, cMET. HGF promotes osteoblastic differentiation of MSCs into bone forming cells, playing a role in bone development, health and repair. AREAS COVERED: This review will focus on the effects of HGF in osteogenesis, bone repair and bone health, including structural and functional insights into the role of HGF in the body. EXPERT OPINION: Approximately 6.2 million Americans experience a fracture annually, with 5-10% being mal- or non-union fractures. HGF is important in priming MSCs for osteogenic differentiation in vitro and is currently being studied to assess its role during bone repair in vivo. Due to the high turnover rate of systemic HGF, non-classic modes of HGF-treatment, including naked-plasmid HGF delivery and the use of HGF splice variants (NK1 & NK2) are being studied to find safe and efficacious treatments for bone disorders, such as mal- or non-union fractures.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b-Mediated Malignant Progression.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.

TAp63γ and ΔNp63ß promote osteoblastic differentiation of human mesenchymal stem cells: regulation by vitamin D3 Metabolites.

The transcription factor p63 is required for skeletal formation, and is important for the regulation of 1α,25(OH)2D3 receptor (VDR) in human mesenchymal stem cells (hMSC). Herein we report that TAp63γ and ΔNp63ß appear to be an integral part of the osteoblastic differentiation of hMSC and are differentially regulated by the vitamin D3 metabolites 1α,25(OH)2D3 and 24R,25(OH)2D3. We compared the endogenous expression of p63 isoforms (TA- and ΔNp63) and splice variants (p63α, -ß, -γ), in naive hMSC and during osteoblastic differentiation of hMSC. TAp63α and -ß were the predominant p63 variants in naive, proliferating hMSC. In contrast, under osteoblastic differentiation conditions, expression of p63 changed from the TAp63α and -ß to the TAp63γ and ΔNp63ß variants. Transient overexpression of the p63 variants demonstrated that TAp63ß, ΔNp63ß, and ΔNp63γ increased alkaline phosphatase activity and ΔNp63α and -γ increased the expression of mRNA for osteocalcin and osterix. Our results support the hypothesis that TAp63α and -ß promote a naive state in hMSC. Moreover, TAp63γ is increased during and promotes early osteoblastic differentiation through the expression of pro-osteogenic genes; VDR, Osterix, Runx2 and Osteopontin. ΔNp63ß also appears to support osteogenic maturation through increased alkaline phosphatase activity. Treatment with 1α,25(OH)2D3 increased the expression of mRNA for ΔNp63, while addition of 24R,25(OH)2D3 increased the expression of TA- and ΔNp63γ variants. These novel findings demonstrate for the first time that p63 variants are differentially expressed in naive hMSC (TAp63α,ß), are important during the osteoblastic differentiation of hMSC (TAp63γ and ΔNp63ß), and are differentially regulated by the vitamin D3 metabolites, 1α,25(OH)2D3 and 24R,25(OH)2D3. The molecular nuances and mechanisms of osteoblastic differentiation presented here will hopefully improve our understanding of bone development, complications in bone repair (mal- and non-union fractures), osteoporosis and possibly lead to new modalities of treatment.

24R,25-dihydroxyvitamin D3 promotes the osteoblastic differentiation of human mesenchymal stem cells.

Although 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is considered the most biologically active vitamin D3 metabolite, the vitamin D3 prohormone, 25-hydroxyvitamin D3 [25(OH)D3], is metabolized into other forms, including 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. Herein we show that 24R,25(OH)2D3 is fundamental for osteoblastic differentiation of human mesenchymal stem cells (hMSCs). Our approach involved analyses of cell proliferation, alkaline phosphatase activity, and pro-osteogenic genes (collagen 1A1, osteocalcin, vitamin D receptor [VDR], vitamin D3-hydroxylating enzymes [cytochrome P450 hydroxylases: CYP2R1, CYP27A1, CYP27B1 and CYP24A1]) and assessment of Ca(2+) mineralization of extracellular matrix. 24R,25(OH)2D3 inhibited hMSC proliferation, decreased 1α-hydroxylase (CYP27B) expression, thereby reducing the ability of hMSCs to convert 25(OH)D3 to 1α,25(OH)2D3, and promoted osteoblastic differentiation through increased alkaline phosphatase activity and Ca(2+) mineralization. 24R,25(OH)2D3 decreased expression of the 1α,25(OH)2D3 receptor, VDR. 24R,25(OH)2D3 but not 1α,25(OH)2D3 induced Ca(2+) mineralization dependent on the absence of the glucocorticoid analog, dexamethasone. To elucidate the mechanism(s) for dexamethasone-independent 1α,25(OH)2D3 inhibition/24R,25(OH)2D3 induction of Ca(2+) mineralization, we demonstrated that 1α,25(OH)2D3 increased whereas 24R,25(OH)2D3 decreased reactive oxygen species (ROS) production. 25(OH)D3 also decreased ROS production, potentially by conversion to 24R,25(OH)2D3. Upon inhibition of the vitamin D3-metabolizing enzymes (cytochrome P450s), 25(OH)D3 increased ROS production, potentially due to its known (low) affinity for VDR. We hypothesize that vitamin D3 actions on osteoblastic differentiation involve a regulatory relationship between 24R,25(OH)2D3 and 1α,25(OH)2D3. These results implicate 24R,25(OH)2D3 as a key player during hMSC maturation and bone development and support the concept that 24R,25(OH)2D3 has a bioactive role in the vitamin D3 endocrine system.

Hepatocyte growth factor and p38 promote osteogenic differentiation of human mesenchymal stem cells.

Hepatocyte growth factor (HGF) is a paracrine factor involved in organogenesis, tissue repair, and wound healing. We report here that HGF promotes osteogenic differentiation through the transcription of key osteogenic markers, including osteocalcin, osterix, and osteoprotegerin in human mesenchymal stem cells and is a necessary component for the establishment of osteoblast mineralization. Blocking endogenous HGF using PHA665752, a c-Met inhibitor (the HGF receptor), or an HGF-neutralizing antibody attenuates mineralization, and PHA665752 markedly reduced alkaline phosphatase activity. Moreover, we report that HGF promotion of osteogenic differentiation involves the rapid phosphorylation of p38 and differential regulation of its isoforms, p38α and p38ß. Western blot analysis revealed a significantly increased level of p38α and p38ß protein, and reverse transcription quantitative PCR revealed that HGF increased the transcriptional level of both p38α and p38ß. Using small interfering RNA to reduce the transcription of p38α and p38ß, we saw differential roles for p38α and p38ß on the HGF-induced expression of key osteogenic markers. In summary, our data demonstrate the importance of p38 signaling in HGF regulation of osteogenic differentiation.

Progerin expression disrupts critical adult stem cell functions involved in tissue repair.

Vascular disease is one of the leading causes of death worldwide. Vascular repair, essential for tissue maintenance, is critically reduced during vascular disease and aging. Efficient vascular repair requires functional adult stem cells unimpaired by aging or mutation. One protein candidate for reducing stem cell?mediated vascular repair is progerin, an alternative splice variant of lamin A. Progerin results from erroneous activation of cryptic splice sites within the LMNA gene, and significantly increases during aging. Mutations triggering progerin overexpression cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS), in which patients die at approximately 13-years of age due to atherosclerosis-induced disease. Progerin expression affects tissues rich in cells that can be derived from marrow stromal cells (MSCs. Studies using various MSC subpopulations and models have led to discrepant results. Using a well-defined, immature subpopulation of MSCs, Marrow Isolated Adult Multilineage Inducible (MIAMI) cells, we find progerin significantly disrupts expression and localization of self-renewal markers, proliferation, migration, and membrane elasticity. One potential treatment, farnesyltransferase inhibitor, ameliorates some of these effects. Our results confirm proposed progerin-induced mechanisms and suggest novel ways in which progerin disturbs critical stem cell functions collectively required for proper tissue repair, offering promising treatment targets for future therapies.

Hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D together stimulate human bone marrow-derived stem cells toward the osteogenic phenotype by HGF-induced up-regulation of VDR.

Bone formation and remodeling require generation of osteoprogenitors from bone marrow stem cells (MSC), which are regulated by growth factors and hormones, with putative roles in mesenchymal cell differentiation. Hepatocyte growth factor (HGF) is a pleiotropic growth factor, and together with its high affinity receptor cMet are widely expressed in normal tissues. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D; produced mainly in the kidney, but also by osteoblasts. We previously reported that HGF and 1,25OHD act together to increase osteogenic differentiation of human MSC (hMSC) potentially through increasing p53. Although p53 does not induce the vitamin D receptor (VDR), p63, a member of the p53 family of transcription factors has been reported to up-regulate VDR expression in some tumor cell lines, and thus might play a part in HGF-regulated VDR expression. Our hypothesis is that the combination of HGF and 1,25OHD can induce hMSC differentiation by up-regulation of 1,25OHD and/or VDR expression to increase cell response(s) to 1,25OHD. Using real-time RT-qPCR, Western blots, luciferase reporter assays, and siRNAs, as well as antibodies to specific signaling molecules we showed that HGF induced VDR gene expression, as well as up-regulated p63 gene expression. p63 gene knockdown by siRNA eliminated the effects of HGF on VDR gene expression as measured by RT-qPCR, Western blots and luciferase reporter assay, and downstream on osteogenic differentiation markers, including alkaline phosphatase staining. Differentiation is a coordinated process of cell cycle exit and tissue-specific gene expression. These results suggest HGF might be a good candidate to coordinate the regulation of these two processes during hMSC osteogenic differentiation. p63 could be a key connecting molecule on the pathway of HGF-induced VDR expression. Understanding the role of these factors and their actions could have important clinical implications for the use of hMSC in the development of novel stem cell therapies.

Human bone marrow-derived stem cell proliferation is inhibited by hepatocyte growth factor via increasing the cell cycle inhibitors p53, p21 and p27.

Human bone marrow-derived stem cells (hMSCs) are a major source of osteoprogenitors. Hepatocyte growth factor (HGF), a glycoprotein constitutively produced by hMSCs, is reported to act on differentiated osteoblasts and also osteoclasts. Moreover, HGF has been shown by us and others to enhance osteoblastic differentiation from hMSCs. Typically, the pro-differentiation effects of HGF have required cooperative action with regulatory factors such as vitamin D or bone matrix material. Here, we have pursued the molecular mechanisms underlying the osteogenic effect of HGF on hMSCs, the principal precursors to bone forming cells. HGF treatment of hMSCs reduced the cell number over time and increased G1/S cell-cycle arrest compared to control (non-treated) cells. RT-qPCR showed treatment with HGF increased gene expression of the cell-cycle inhibitors p53, p21, and p27, possibly explaining the cell growth inhibition and G1 arrest, a step critical to phenotypic differentiation. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect on hMSCs and the HGF-mediated increase in p53, p21, and p27, strongly supporting a role for these cell-cycle inhibitors in HGF's regulation of hMSCs. HGF in combination with a known inducer of osteogenic differentiation, 1,25-dihydroxyvitamin D, significantly increased cell maturation/differentiation as indicated by an increase in several osteoblast markers. Taken together these results demonstrate that HGF significantly enhances hMSC osteoblast differentiation by 1,25-dihydroxyvitamin D.

Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers.

Cell-based therapies for global cerebral ischemia represent promising approaches for neuronal damage prevention and tissue repair promotion. We examined the potential of marrow-isolated adult multilineage-inducible (MIAMI) cells, a homogeneous subpopulation of immature human mesenchymal stromal cell, injected into the hippocampus to prevent neuronal damage induced by global ischemia using rat organotypic hippocampal slices exposed to oxygen-glucose deprivation and rats subjected to asphyxial cardiac arrest. We next examined the value of combining fibronectin-coated biomimetic microcarriers (FN-BMMs) with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) pre-treated MIAMI compared to EGF/bFGF pre-treated MIAMI cells alone, for their in vitro and in vivo neuroprotective capacity. Naïve and EGF/bFGF pre-treated MIAMI cells significantly protected the Cornu Ammonis layer 1 (CA1) against ischemic death in hippocampal slices and increased CA1 survival in rats. MIAMI cells therapeutic value was significantly increased when delivering the cells complexed with FN-BMMs, probably by increasing stem cell survival and paracrine secretion of pro-survival and/or anti-inflammatory molecules as concluded from survival, differentiation and gene expression analysis. Four days after oxygen and glucose deprivation and asphyxial cardiac arrest, few transplanted cells administered alone survived in the brain whereas stem cell survival improved when injected complexed with FN-BMMs. Interestingly, a large fraction of the transplanted cells administered alone or in complexes expressed ßIII-tubulin suggesting that partial neuronal transdifferentiation may be a contributing factor to the neuroprotective mechanism of MIAMI cells.

Changes in the expression of genes associated with intraneuronal amyloid-beta and tau in Alzheimer's disease.

The clinical hallmark of Alzheimer's disease (AD) is impairment of cognition associated with loss of synapses, accumulation of amyloid-beta (Abeta) both within neurons and as extracellular deposits, and neurofibrillary degeneration composed of phospho-tau. Neurons in the hippocampus are among those that are most vulnerable. The purpose of this study was to investigate the expression of genes associated with cognition, synapse, and mitochondrial function in hippocampal neurons of AD compared to normal individuals. Neurons from the hippocampus with intraneuronal Abeta immunoreactivity were captured with laser microdissection; RNA was extracted; and levels of brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), dynamin-1 (DYN), and cytochrome C oxidase subunit II (COX2) were assessed with quantitative real-time polymerase chain reaction. We found no significant differences in the expression of these genes in AD between neurons associated with Abeta compared to those lacking Abeta immunoreactivity. Double immunofluorescence microscopy showed the number of hippocampal neurons coexpressing Abeta or phospho-tau and either BDNF, TrkB, or DYN was similar in AD and controls. Our results suggest that neither intraneuronal Abeta nor phospho-tau has obligatory effects on reducing the expression of genes important for memory and cognition in hippocampus of AD.

Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells.

We recently reported the isolation of a unique subpopulation of human stromal cells from bone marrow (BM) termed marrow-isolated adult multilineage inducible (MIAMI) cells, capable of differentiating in vitro into mature-like cells from all three germ layers. The oxygen tension (pO2) in BM ranges from 1 to 7%, which prompted us to examine the role of pO2 in regulating the capacity of MIAMI cells both to self-renew and maintain their pluripotentiality (stemness) or to progress toward osteoblastic differentiation. MIAMI cells were grown under low-pO2 conditions (1, 3, 5, and 10% oxygen) or air (21% oxygen). The proliferation rate of cells exposed to 3% oxygen (3 days) increased, resulting in cell numbers more than threefold higher than those of cells exposed to air (at 7 days). In cells grown under osteoblastic differentiation conditions, the expression of the osteoblastic markers osteocalcin, bone sialoprotein, osterix, and Runx2 and alkaline phosphatase activity was upregulated when incubated in air; however, it was blocked at low (3%) pO2. Similarly, biomineralization of long-term cell cultures was high under osteoblastic differentiation conditions in air but was undetectable at low (3%) pO2. In contrast, low pO2 upregulated mRNAs for OCT-4, REX-1, telomerase reverse transcriptase, and hypoxia-inducible factor-1 alpha, and increased the expression of SSEA-4 compared to air. Moreover, the expression of embryonic stem cell markers was sustained even under osteogenic culture conditions. Similar results were obtained using commercially available marrow stromal cells. We hypothesize a physiological scenario in which primitive MIAMI cells self-renew while localized to areas of low pO2 in the bone marrow, but tend to differentiate toward osteoblasts when they are located closer to blood vessels and exposed to higher pO2. Our results strongly suggest that maintaining developmentally primitive human cells in vitro at low pO2 would be more physiological and favor stemness over differentiation.

Sustained stromal stem cell self-renewal and osteoblastic differentiation during aging.

We have reported the isolation of a unique subpopulation of human stromal cells from bone marrow termed marrow-isolated adult multilineage inducible (MIAMI) cells. The expression of embryonic stem cell markers SSEA-4, Oct-4, Rex-1, and telomerase reverse transcriptase indicates the developmentally immature status of these cells. They resemble primitive stem cells in their capacity to differentiate, at least in vitro, into mature-like cells from all three germ layers. MIAMI cells are characterized by a unique molecular profile that distinguishes them from other marrow stromal cell populations. Although the frequency of MIAMI cells, among all marrow nucleated cells, decreases from 0.01% at age 3 to 0.0018% at age 45, their numbers remain unchanged after age 45. The level of expression of the markers characteristic of MIAMI cells remains constant independent of age and gender. In long-term in vitro expansion experiments aging increased the population doubling time by about 30%, whereas specific in vitro differentiation of MIAMI cells toward osteoblastic cells was unaffected. Because the oxygen tension in bone marrow ranges from 1% to 7%, we examined the role of oxygen tension in regulating the capacity of MIAMI cells to self-renew and maintain their pluripotentiality during long-term culture. Low oxygen tension upregulated mRNAs for primitive embryonic stem cell markers. Our results suggest that maintaining developmentally primitive human cells in vitro at low oxygen tension is more physiologic and favors stemness. For osteoblastic differentiation, gap-junctional communication mediated by connexin43 is required. Its inhibition not only blocked osteoblastic differentiation but stimulated the adipocytic differentiation.

Androgen-induced mineralization by MC3T3-E1 osteoblastic cells reveals a critical window of hormone responsiveness.

Despite their clinical importance for skeletal growth and homeostasis, the actions of androgens on osteoblastic cells are not well understood. MC3T3-E1 cells, a nontransformed murine preosteoblastic cell line, that traverse the stages of osteoblastic differentiation within 30 days in vitro, were exposed to mibolerone (an androgen receptor (AR) agonist) or 5alpha-dihydroxytestosterone (DHT) from days 3 to 30 post-plating. Cells exposed to this hormonal regimen exhibited a significant increase in mineralization (calcium deposition) compared to vehicle-treated cells. Delaying treatment for 4-11 days (treatment still completed on day 30 post-plating) enhanced mineralization further. Within 2 days post-plating, AR protein increased 7.2-fold in androgen-treated cells and 2.5-fold in vehicle-treated cells. MC3T3-E1 cells transfected with an androgen- and glucocorticoid-responsive reporter construct on day 1 post-plating followed by a 2 day exposure to DHT, mibolerone, or dexamethasone (dex; a glucocorticoid receptor agonist) exhibited reporter gene activation only with dex treatment. In contrast, delaying transfection and treatment for at least 1 day resulted in comparable androgen- and dex-mediated reporter gene transactivation. Therefore, the ability of MC3T3-E1 cells to respond to androgens is dependent on the timing of androgen administration.

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.

Autosomal dominant hyperostosis/osteosclerosis with high serum alkaline phosphatase activity.

We studied eight affected and four unaffected individuals from a Colombian family with autosomal dominant diffuse high bone density. Affected individuals have normal, proportional height and high serum alkaline phosphatase activity. Radiographically, affected members exhibit generalized, symmetrically diffuse endosteal hyperostosis of the long bones and skull with narrow medullary cavities and loss of the diploë, respectively. There is no periosteal reaction or decreased hematopoiesis. Furthermore, osteosclerosis affects vertebral bodies, ribs, pelvis, mandible, clavicles, and scapulae. Bone mineral density is 2.4-7.3 SD above the mean for age and gender in affected individuals. Affected vs. unaffected individuals' Z-scores were (mean +/- SD) 5.03 +/- 1.77 vs. 0.08 +/- 0.97, respectively, P = 0.0004). Three affected subjects older than 40 yr old lost bone mass in 6 yr. No dysmorphism, abnormal facial features, bone fractures, or cranial nerve involvement was found. The pattern of inheritance, the absence of asymmetries and malformations, the increased serum alkaline phosphatase, the peak bone mass that appears to decrease physiologically with age, and the involvement of cortical and trabecular bone suggest a new variant of hyperostosis/osteosclerosis that affects the entire skeleton.

The G gamma / T-15 transgenic mouse model of androgen-independent prostate cancer: target cells of carcinogenesis and the effect of the vitamin D analogue EB 1089.

Transgenic mouse models of prostate cancer provide unique opportunities to understand the molecular events in prostate carcinogenesis and for the preclinical testing of new therapies. We studied the G gamma T-15 transgenic mouse line, which contains the human fetal globin promoter linked to SV40 T antigen (Tag) and which develops androgen-independent prostate cancer. Using the immunohistochemistry of normal mouse prostates before tumor formation, we showed that the target cells of carcinogenesis in G gamma T-15 mice are located in the basal epithelial layer. We tested the efficacy of the 1,25(OH)(2)D(3) analogue, EB 1089, to chemoprevent prostate cancer in these transgenic mice. Compared with treatment with placebo, treatment with EB 1089 at three different time points before the onset of prostate tumors in mice did not prevent or delay tumor onset. However, EB 1089 significantly inhibited prostate tumor growth. At the highest dose, EB 1089 inhibited prostate tumor growth by 60% (P = 0.0003) and the growth in the number of metastases, although this dose also caused significant hypercalcemia and weight loss. We conducted several in vitro experiments to explore why EB 1089 did not prevent the occurrence of the primary tumors. EB 1089 significantly inhibited the growth of a Tag-expressing human prostate epithelial cell line, BPH-1, and an androgen-insensitive subline of LNCaP cells [which was not inhibited by 1,25(OH)(2)D(3)]. Thus, neither Tag expression nor androgen insensitivity explain the absence of chemopreventive effect. Conversely, neither 1,25(OH)(2)D(3) nor EB 1089 inhibited the growth of the normal rat prostate basal epithelial cell line NRP-152. It is likely that EB 1089 was not effective in delaying the growth of the primary tumor in G gamma T-15 transgenic mice because the target cells of carcinogenesis in these mice are located in the basal epithelial layer. We conclude that G gamma T-15 transgenic mice are a useful model for testing vitamin D-based therapies in androgen-insensitive prostate cancer but are not suitable for studies of vitamin D-based chemoprevention. The superiority of EB 1089 over 1,25(OH)(2)D(3) in the growth suppression of androgen-insensitive prostate cancer cells supports the use of EB 1089 in androgen-insensitive prostate cancer.