Paul C. Schiller

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.

Adult cell therapy for brain neuronal damages and the role of tissue engineering

No long term effective treatments are currently available for brain neurological disorders such as stroke/cerebral ischemia, traumatic brain injury and neurodegenerative disorders. Cell therapy is a promising strategy, although alternatives to embryonic/foetal cells are required to overcome ethical, tissue availability and graft rejection concerns. Adult cells may be easily isolated from the patient body, therefore permitting autologous grafts to be performed. Here, we describe the use of adult neural stem cells, adrenal chromaffin cells and retinal pigment epithelium cells for brain therapy, with a special emphasis on mesenchymal stromal cells. However, major problems like cell survival, control of differentiation and engraftment remain and may be overcome using a tissue engineering strategy, which provides a 3D support to grafted cells improving their survival. New developments, such as the biomimetic approach which combines the use of scaffolds with extracellular matrix molecules, may improve the control of cell proliferation, survival, migration, differentiation and engraftment in vivo. Therefore, we later discuss scaffold properties required for brain cell therapy as well as new tissue engineering advances that may be implemented in combination with adult cells for brain therapy. Finally, we describe an approach developed in our laboratory to repair/protect lesioned tissues: the pharmacologically active microcarriers.

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

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.

Mesenchymal stem cells as cellular vehicles for delivery of nanoparticles to brain tumors

The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemo-therapies. Nanotechnologies represent great promise in glioma therapy as they protect therapeutic agent and allow its sustained release. However, new paradigms allowing tumor specific targeting and extensive intratumoral distribution must be developed to efficiently deliver nanoparticles (NPs). Knowing the tropism of mesenchymal stem cells (MSCs) for brain tumors, the aim of this study was to obtain the proof of concept that these cells can be used as NP delivery vehicles. Two types of NPs loaded with coumarin-6 were investigated: poly-lactic acid NPs (PLA-NPs) and lipid nanocapsules (LNCs). The results show that these NPs can be efficiently internalized into MSCs while cell viability and differentiation are not affected. Furthermore, these NP-loaded cells were able to migrate toward an experimental human glioma model. These data suggest that MSCs can serve as cellular carriers for NPs in brain tumors.

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.

The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats.

Multipotent mesenchymal stromal cells (MSCs) raise great interest for brain cell therapy due to their ease of isolation from bone marrow, their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells and to secrete a variety of growth factors and chemokines. In this study, we assessed the effects of a subpopulation of human MSCs, the marrow-isolated adult multilineage inducible (MIAMI) cells, combined with pharmacologically active microcarriers (PAMs) in a rat model of Parkinsons disease (PD). PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) microspheres, coated by a biomimetic surface and releasing a therapeutic protein, which acts on the cells conveyed on their surface and on their microenvironment. In this study, PAMs were coated with laminin and designed to release neurotrophin 3 (NT3), which stimulate the neuronal-like differentiation of MIAMI cells and promote neuronal survival. After adhesion of dopaminergic-induced (DI)-MIAMI cells to PAMs in vitro, the complexes were grafted in the partially dopaminergic-deafferented striatum of rats which led to a strong reduction of the amphetamine-induced rotational behavior together with the protection/repair of the nigrostriatal pathway. These effects were correlated with the increased survival of DI-MIAMI cells that secreted a wide range of growth factors and chemokines. Moreover, the observed increased expression of tyrosine hydroxylase by cells transplanted with PAMs may contribute to this functional recovery.

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.

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

Previously we showed that physiological levels of parathyroid hormone (PTH) can increase the mineralization of extracellular matrix (ECM) by osteoblast-like cells in vitro. In this study, we assess the role of gap-junctional intercellular communication (GJC) in the PTH-enhanced mineralization of ECM in MC3T3-E1 cells, a murine culture model of osteoblastic differentiation. Messenger RNA and protein for connexin 43 (Cx43), the major component of MC3T3-E1 gap junctions, and GJC increased as the cells progressed toward a mature phenotype. Immunocytochemistry showed accumulation of Cx43 at the area of close contact between cells. The timing of the PTH treatment that increased matrix mineralization in these cells coincided with the highest expression of Cx43 and GJC. Administration of 18-alpha-glycyrrhetinic acid (AGA) promptly blocked GJC in cultures of MC3T3-E1 cells in a dose-dependent and reversible manner at all times tested during the culture period. Treatment with AGA, but not with an inactive analog, reversed the PTH-induced ECM mineralization. These data suggest that GJC mediates anabolic actions of PTH related to osteoblast-mediated mineralization.