Gianluca D'Ippolito

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.

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

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.

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

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.

Induction of COX-2 and reactive gliosis by P2Y receptors in rat cortical astrocytes is dependent on ERK1/2 but independent of calcium signalling.

The present study has been aimed at characterizing the ATP/P2 receptor (and transductional pathways) responsible for the morphological changes induced in vitro by αβmethyleneATP on rat astrocytes obtained from cerebral cortex, a brain area highly involved in neurodegenerative diseases. Exposure of cells to this purine analogue resulted in elongation of cellular processes, an event reproducing in vitro a major hallmark of in vivo reactive gliosis. αβmethyleneATP‐induced gliosis was prevented by the P2X/P2Y blocker pyridoxalphosphate‐6‐azophenyl‐2′‐4′‐disulfonic acid, but not by the selective P2X antagonist 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP, ruling out a role for ligand‐gated P2X receptors. Conversely, the Gi/Go protein inactivator pertussis toxin completely prevented αβmethyleneATP‐induced effects. No effects were induced by αβmethyleneATP on intracellular calcium concentrations. RT‐PCR and western blot analysis showed that αβmethyleneATP–induced gliosis involves up‐regulation of cyclooxygenase‐2 (but not lipooxygenase). Also this effect was fully prevented by pyridoxalphosphate‐6‐azophenyl‐2′‐4′‐disulfonic acid. Experiments with inhibitors of mitogen‐activated protein kinases (MAPK) suggest that extracellular signal regulated protein kinases (ERK)1/2 mediate both cyclooxygenase‐2 induction and the associated in vitro gliosis. These findings suggest that purine‐induced gliosis involves the activation of a calcium‐independent G‐protein‐coupled P2Y receptor linked to ERK1/2 and cyclooxygenase‐2. Based on the involvement of cyclooxygenase‐2 and inflammation in neurodegenerative diseases, these findings open up new avenues in the identification of novel biological targets for the pharmacological manipulation of neurodegeneration.

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

J. Neurochem. (2011) 119, 972–988.

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 HGFs 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.

Human marrow-isolated adult multilineage-inducible (MIAMI) cells protect against peripheral vascular ischemia in a mouse model

BACKGROUND AIMS The treatment of peripheral vascular disease (PVD) with stem cells potentially offers a promising strategy. We tested marrow-isolated adult multilineage-inducible (MIAMI) cells to induce neovascularization in a mouse model of critical hindlimb ischemia (CLI). METHODS CLI was induced in the right hindlimb of Balb/C mice. One million MIAMI cells, normally grown at 3% O₂, were injected in the adductor muscle along the ischemic region. All animals (n = 11 per group) were immunosuppressed with cyclosporine daily for the entire period. Human foreskin fibroblast (HFF) cells and phosphate-buffered saline (PBS) were used as controls. Blood perfusion in the ischemic right and non-ischemic left hindlimbs was measured. RESULTS Compared with animals receiving HFF cells or PBS, MIAMI cells significantly improved blood perfusion, necrosis and inflammation in the ischemic limb. A fraction of injected MIAMI cells expressed CD31 and von Willebrand factor (vWF). MIAMI cells in vitro, under pro-angiogenic growth conditions, differentiated into endothelial-like cells and expressed endothelial markers such as CD31 and vWF, determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and CD31 and kinase insert domain receptor (KDR), determined by immunofluorescence. Moreover, MIAMI cells formed vascular endothelial-like tubules in the presence of matrigel. Bioplex immunoassay analysis showed increased secretion of angiogenic/anti-inflammatory factors by the MIAMI cells under 3% O₂ compared with 21% O₂, including monocyte chemoattractant protein-1 (MCP-1), fractalkine (Ftk), growth-related oncogene (GRO), vascular endothelial growth factor (VEGF), interleukin (IL)-6 and IL-8. Furthermore, transcripts for anti-inflammatory molecules stanniocalcin-1 (STC-1) and tumor necrosis factor-α-stimulated gene 6 (TSG-6) were up-regulated several fold. CONCLUSIONS MIAMI cells can be very useful for patients affected by CLI. MIAMI cells promote blood vessel formation and reduce inflammation and necrosis in ischemic tissue.

Epigenetic regulation of embryonic stem cell marker miR302C in human chondrosarcoma as determinant of antiproliferative activity of proline-rich polypeptide 1

Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. We described recently that tumor growth inhibiting cytostatic proline-rich polypeptide 1, (PRP-1) significantly upregulated tumor suppressor miRNAs, downregulated onco-miRNAs in human chondrosarcoma JJ012 cell line, compared to chondrocytes culture. In this study we hypothesized the existence and regulation of a functional marker in cancer stem cells, correlated to peptides antiproliferative activity. Experimental results indicated that among significantly downregulated miRNA after PRP-1treatment was miRNAs 302c*. This miRNA is a part of the cluster miR302-367, which is stemness regulator in human embryonic stem cells and in certain tumors, but is not expressed in adult hMSCs and normal tissues. PRP-1 had strong inhibitory effect on viability of chondrosarcoma and multilineage induced multipotent adult cells (embryonic primitive cell type). Unlike chondrosarcoma, in glioblastoma, PRP-1 does not have any inhibitory activity on cell proliferation, because in glioblastoma miR-302-367 cluster plays an opposite role, its expression is sufficient to suppress the stemness inducing properties. The observed correlation between the antiproliferative activity of PRP-1 and its action on downregulation of miR302c explains the peptides opposite effects on the upregulation of proliferation of adult mesenchymal stem cells, and the inhibition of the proliferation of human bone giant-cell tumor stromal cells, reported earlier. PRP-1 substantially downregulated the miR302c targets, the stemness markers Nanog, c-Myc and polycomb protein Bmi-1. miR302c expression is induced by JMJD2-mediated H3K9me2 demethylase activity in its promoter region. JMJD2 was reported to be a positive regulator for Nanog. Our experimental results proved that PRP-1 strongly inhibited H3K9 activity comprised of a pool of JMJD1 and JMJD2. We conclude that inhibition of H3K9 activity by PRP-1 leads to downregulation of miR302c and its targets, defining the PRP-1 antiproliferative role.

Marrow-isolated adult multilineage inducible cells embedded within a biologically-inspired construct promote recovery in a mouse model of peripheral vascular disease

Peripheral vascular disease is one of the major vascular complications in individuals suffering from diabetes and in the elderly that is associated with significant burden in terms of morbidity and mortality. Stem cell therapy is being tested as an attractive alternative to traditional surgery to prevent and treat this disorder. The goal of this study was to enhance the protective and reparative potential of marrow-isolated adult multilineage inducible (MIAMI) cells by incorporating them within a bio-inspired construct (BIC) made of two layers of gelatin B electrospun nanofibers. We hypothesized that the BIC would enhance MIAMI cell survival and engraftment, ultimately leading to a better functional recovery of the injured limb in our mouse model of critical limb ischemia compared to MIAMI cells used alone. Our study demonstrated that MIAMI cell-seeded BIC resulted in a wide range of positive outcomes with an almost full recovery of blood flow in the injured limb, thereby limiting the extent of ischemia and necrosis. Functional recovery was also the greatest when MIAMI cells were combined with BICs, compared to MIAMI cells alone or BICs in the absence of cells. Histology was performed 28 days after grafting the animals to explore the mechanisms at the source of these positive outcomes. We observed that our critical limb ischemia model induces an extensive loss of muscular fibers that are replaced by intermuscular adipose tissue (IMAT), together with a highly disorganized vascular structure. The use of MIAMI cells-seeded BIC prevented IMAT infiltration with some clear evidence of muscular fibers regeneration.