Marrow-isolated Adult Multilineage Inducible Cells Research Articles

Effect of Stem Cell Augmented Micronized Allograft on Arthrodesis in the Ankle and Foot

Category: Basic Sciences/Biologics Introduction/Purpose: Both primary and revision arthrodesis are mainstays of foot and ankle surgery. Many studies have investigated the effect of autograft, varying forms of bone graft substitutes, and of biologic agents on bone healing, including in the setting of arthrodesis. A commercially available stem cell augmented micronized allograft (Via Graft®) is comprised of micronized (100-300 microns) allograft bone with marrow-isolated adult multilineage inducible cells (MIAMI cells). It has been proposed as an alternative to autograft and as an adjunct to arthrodesis. We present a series of 86 patients with minimum one-year follow-up who underwent either primary or revision arthrodesis with Via Graft®, with or without additional graft. Methods: Between 2015 and 2016, 86 patients treated with Via Graft® were identified with minimum one-year follow-up. Data was collected including demographic information, BMI, smoking history, medical comorbidities; indication for fusion (including Charcot arthropathy, posttraumatic arthritis, rheumatoid arthritis, AVN, congenital anomalies, neuromuscular disorders, revision surgery, post-infectious, previous total ankle arthroplasty, posterior tibial tendon dysfunction); procedure performed (ankle, hindfoot, midfoot, or forefoot fusion), number of joints fused, use of additional graft, radiographic union, and complications. Fusion was evaluated based upon radiographic or CT evidence of osseous bridging. The unit of the analysis was the joint. Generalized Linear Mixed Model (GLMM) was utilized to evaluate the association between fusion and the data set described above. Statistical analyses were performed using SAS® 9.4. Results: 191 joints were fused in 86 patients (F: 48, M: 38). Mean age was 58.7 years, mean follow-up was 15.8 months. Radiographic union was achieved in 163/191 (85.3%) joints. Age, gender, smoking status, presence of diabetes, history of Charcot, revision fusion, or use of cancellous autograft or allograft demonstrated no statistically significant differences in fusion rates. Intramedullary talocalcaneal arthrodesis (IMTCA), with or without additional joints fused simultaneously (p=.001), use of structural allograft (most commonly femoral head)(p<.001), structural autograft (p=.001), history of previous TAA (p<.0001), and history of AVN (p=.042) demonstrated statistically significant rates of nonunion, despite use of Via Graft® (Table 1). Conclusion: This study is the largest series to date of Via Graft® augmentation of arthrodesis. Lower union rates were found to be associated with patients undergoing hindfoot fusion with an IMTCA with or without additional joints fused, a history of failed total ankle replacement or AVN of the talus, and use of femoral head allograft. However, this preliminary study requires further analysis given the difficult and salvage nature of these cases. As bony union is multifactorial, the selection of bone graft and biologic agents is an important factor, but may not be sufficient to overcome the biology of the host.

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.

Pharmacologically active microcarriers delivering BDNF within a hydrogel: Novel strategy for human bone marrow-derived stem cells neural/neuronal differentiation guidance and therapeutic secretome enhancement.

Combinatorial tissue engineering strategies for the central nervous system are scarce. We developed and characterized a novel injectable non-toxic stem cell and protein delivery system providing regenerative cues for central nervous system disorders. BDNF, a neurotrophic factor with a wide-range effect, was nanoprecipitated to maintain its structure and released in a sustained manner from novel polymeric microcarriers. The combinatorial 3D support, provided by fibronectin-microcarriers and the hydrogel, to the mesenchymal stem cells guided the cells towards a neuronal differentiation and enhanced their tissue repair properties by promoting growth factors and cytokine secretion. The long-term release of physiological doses of bioactive BDNF, combined to the enhanced secretion of tissue repair factors from the stem cells, constitute a promising therapeutic approach.

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 β-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.

Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease.

Stem cell-based regenerative therapies hold great potential for the treatment of degenerative disorders such as Parkinson's disease (PD). We recently reported the repair and functional recovery after treatment with human marrow-isolated adult multilineage inducible (MIAMI) cells adhered to neurotrophin-3 (NT3) releasing pharmacologically active microcarriers (PAMs) in hemiparkinsonian rats. In order to comprehend this effect, the goal of the present work was to elucidate the survival, differentiation, and neuroprotective mechanisms of MIAMI cells and human neural stem cells (NSCs), both adhering to NT3-releasing PAMs in an ex vivo organotypic model of nigrostriatal degeneration made from brain sagittal slices. It was shown that PAMs led to a marked increase in MIAMI cell survival and neuronal differentiation when releasing NT3. A significant neuroprotective effect of MIAMI cells adhering to PAMs was also demonstrated. NSCs barely had a neuroprotective effect and differentiated mostly into dopaminergic neuronal cells when adhering to PAM-NT3. Moreover, those cells were able to release dopamine in a sufficient amount to induce a return to baseline levels. Reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses identified vascular endothelial growth factor (VEGF) and stanniocalcin-1 as potential mediators of the neuroprotective effect of MIAMI cells and NSCs, respectively. It was also shown that VEGF locally stimulated tissue vascularization, which might improve graft survival, without excluding a direct neuroprotective effect of VEGF on dopaminergic neurons. These results indicate a prospective interest of human NSC/PAM and MIAMI cell/PAM complexes in tissue engineering for PD. Stem cell-based regenerative therapies hold great potential for the treatment of degenerative disorders such as Parkinson's disease (PD). The present work elucidates and compares the survival, differentiation, and neuroprotective mechanisms of marrow-isolated adult multilineage inducible cells and human neural stem cells both adhered to neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo organotypic model of PD made from brain sagittal slices.

A plethora of human pluripotent stem cells

At the early stages of mammalian development, a number of developmentally plastic cells appear that possess the ability to give rise to all of the differentiated cell types normally derived from the three primary germ layers - unique character known as pluripotency. To date, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been shown to be truly pluripotent. However, recent studies have revealed a variety of other cells that demonstrate pluripotentiality, including very small embryonic-like stem cells (VSELs), amniotic fluid stem cells (AFSCs), marrow-isolated adult multilineage inducible cells (MIAMI) and multipotent adult precursor cells (MAPCs). This review summarises key features of these six kinds of pluripotent and potentially pluripotent stem cells (ESCs, iPSCs, VSELs, AFSCs, MIAMI and MAPCs) and the evidence for their pluripotency properties.

In vitro and in vivo interactions between glioma and marrow-isolated adult multilineage inducible (MIAMI) cells

The prognosis of patients with malignant glioma remains extremely poor despite surgery and improvements in radio- and chemo-therapies. We recently showed that marrow-isolated adult mutilineage inducible (MIAMI) cells, a subpopulation of human mesenchymal stromal cells (MSCs), can serve as cellular carriers of drug-loaded nanoparticles to brain tumors. However, the safety of MIAMI cells as cellular treatment vectors in glioma therapy must be evaluated, in particular their effect on glioma growth and their fate in a tumor environment. In this study, we showed that MIAMI cells were able to specifically migrate toward the orthotopic U87MG tumor model and did not influence its growth. In this model, MIAMI cells did not give rise to cells resembling endothelial cells, pericytes, cancer-associated fibroblasts (CAFs), or astrocytes. Despite these encouraging results, the effects of MIAMI cells may be glioma-dependent. MIAMI cells did not migrate toward the orthotopic Lab1 GB and they can induce the proliferation of other glioma cell lines in vitro. Furthermore, a fraction of MIAMI cells was found to be in a state of proliferation in the U87MG tumor environment. These findings indicate that the use of MIAMI cells as cellular treatment vectors for malignant tumors must be controlled. These cells may be used as “suicide vectors”: vectors for killing not only tumor cells but themselves.

Comparative analysis of protein expression of three stem cell populations: Models of cytokine delivery system in vivo

Several mechanisms mediate the regenerative and reparative capacity of stem cells, including cytokine secretion; therefore these cells can act as delivery systems of therapeutic molecules. Here we begin to address the molecular and cellular basis of their regenerative potential by characterizing the proteomic profile of human embryonic stem cells (hESCs), mesenchymal stem cells (hMSCs) and marrow isolated adult multilineage inducible (MIAMI) cells, followed by analysis of the secretory profile of the latter stem cell population. Proteomic analysis establishes the closer relationship between hMSCs and MIAMI cells, while hESCs are more divergent. However, MIAMI cells appear to have more proteins in common with hESCs than hMSCs. Proteins characteristic of hMSCs include transgelin-2, phosphatidylethanolamine-binding protein 1 (PEBP1), Heat-Shock 20 kDa protein (HSP20/HSPβ6), and programmed cell death 6-interacting protein (PDC6I) among others. MIAMI cells are characterized by the high level expression of ubiquitin carboxyl-terminal hydrolase isoenzyme L1 (UCHL1), 14-3-3 zeta, HSP27 (HSPβ1), and tropomyosin 4 and 3. For hESC, elongation factor Tu (EFTu), isocitrate dehydrogenase (IDH1) and the peroxiredoxins 1, 2, and 6 (PRDX1, PRDX2, and PRDX6) were the most characteristic. Secretome analysis indicates that MIAMI cells secrete higher levels of vascular endothelial growth factor (VEGF), Fractalkine, Interleukin-6, interlukin-8, and growth related oncogene (GRO), compared to hMSCs. These soluble mediators are known to play key roles in angiogenesis, arteriogenesis, atheroprotection, immunomodulation, neuroprotection, axonal growth, progenitor cell migration, and prevention of apoptosis. All these roles are consistent with a reparative pro-survival secretory phenotype. We further discuss the potential of these cells as therapeutic vehicles.

Rac1b regulates NT3-stimulated Mek-Erk signaling, directing marrow-isolated adult multilineage inducible (MIAMI) cells toward an early neuronal phenotype

Due to the limitations of neural stem cells to repair neuronal damage in the human brain, alternative approaches of repair using autologous adult stem cells have been examined for direct cell-replacement, or paracrine mediated neuroprotective effects. Human bone marrow–derived stromal cells (hMSCs) are a heterogeneous adult stem cell population with diverse immunomodulatory properties and the potential to differentiate into cells characteristic of all three germ layers. hMSCs are a renewable source of progenitor cells suitable for cell-based tissue repair. The marrow isolated adult multilineage inducible (MIAMI) cells developed by our laboratory are a developmentally immature homogeneous subpopulation of hMSCs that maintain self-renewal potential during ex vivo expansion, efficient differentiation capacity into neuron-like cells in vitro, as well as direct in vivo neuroprotection and functional recovery in animal models of neurological diseases.We now address the early signaling mechanisms regulating the neuron-like differentiation of MIAMI cells in vitro, in response to activation of the neurotrophic tyrosine–kinase receptor, type 3 (NTRK3) via neurotrophin 3 (NT3). We molecularly characterize a novel role for Rac1b mediating the neurogenic potential of MIAMI cells. Rac1b had an overall negative modulatory effect on the NT3-stimulated Mek1/2-Erk1/2 signaling pathway, proneuronal gene expression and neurite-like extensions. Rac1b was required for NT3-stimulated cell proliferation of MIAMI cells, yet was found to repress CCND1 and CCNB1 mRNA expression independent of NT3 stimulation, suggesting a dual neurotrophin dependent/independent function. Differential levels of Rac1b activity in hMSCs may explain the apparent contradictory reports regarding their neurogenic potential. These findings demonstrate the in vitro neurogenic potential of hMSCs as governed by Rac1b during NT3 stimulation.

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 βIII-tubulin suggesting that partial neuronal transdifferentiation may be a contributing factor to the neuroprotective mechanism of MIAMI cells.

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

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). 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. 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. 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.

Multiple strategies must be pursued in the development of cellular therapies

Stem cell-based therapies are at the forefront of research into novel ways to treat many disease processes. However, providing clinically useful diseasespecifi c cellular therapies will require further pre-clinical work. Discovery of new cell lines, development of improved pre-transplantation processing protocols, and further research into the mechanisms behind non-regenerative and regenerative healing are among the important factors involved in driving this fi eld to clinical application (1). The articles in this issue of Cytotherapy provide examples of the above strategies, as applied to peripheral vascular disease, myocardial ischemia and cutaneous wound healing, and highlight the vital role of neo-angiogenesis in the regenerative healing process. In their article, Rahnemai-Azar et al . (2) introduce human marrow-isolated adult multilineage-inducible (MIAMI) cells. When grown at low oxygen tension (3% O 2 ), these cells are capable of differentiation into endothelial cells and produce high levels of proangiogenic factors, including monocyte chemoattractant protein 1 (MCP-1), fractalkine, growth-related oncogene (GRO), interleukin-6 (IL-6), IL-8 and vascular endothelial growth factor (VEGF). Transplantation of these cells into the ischemic hindlimbs of mice signifi cantly reduced limb ischemia and necrosis and improved perfusion compared with fi broblast and vehicle controls. Immunohistochemistry demonstrated that surviving MIAMI cells were associated with blood vessels or the perivascular space, and a portion expressed CD31 and/or von Willebrand factor (vWF). While further work needs to be done, and comparisons made with other endothelial progenitor cell populations, MIAMI cells appear to be a promising cell population in promoting tissue reperfusion and angiogenesis. Huang et al . (3) present their research into human umbilical cord lining epithelial cells (CLEC) as a potential source of skin-type epithelial stem cells. CLECs possess many stem cell characteristics, including long telomere lengths, high proliferative potential and expression of the pluripotent markers OCT-4, SOX2 and Nanog, but in small proportions. CLECs also have many cytokeratin markers usually specifi c to basal keratinocytes, such as CK8 and CK14, as well as p63 and CK19, markers for epidermal skin progenitor cells. In organotypic culture, these cells formed a stratifi ed epithelium typical of skin, but they failed to form a cornifi ed strata layer. CLECs have limitations, as discussed in the article; however, there is potential in applications for promoting wound healing and skin regeneration if these limitations can be addressed. For the treatment of ischemic disease processes, stem cells must fi rst home to the area of insult and then overcome the high oxidative stress of the ischemic tissue (4). Activation of the PI3/Akt pathway is believed to provide protection against apoptosis induced by oxidative stress. Zhang et al . (5) report that zinc supplementation (10 μ mol/mL) of the growth media of bone marrow-derived mesenchymal stromal cells (MSC) reduced apoptosis, enhanced VEGF production and was associated with increased levels of Akt phosphorylation in vitro . Transplantation of zinc-treated MSC into murine ischemic hindlimbs resulted in increased cell survival, angiogenesis and reperfusion compared with untreated MSC. Strategies to improve the survival of transplanted cells in hostile environments are essential in the development of cellular-based therapies. The article by Lee et al . (6) highlights the potential paracrine effects following stem cell therapy. Repeated Cytotherapy, 2011; 13: 133–134

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 Parkinson’s 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.

EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells

Aims Multipotent mesenchymal stromal cells raise great interest for regenerative medicine studies. Some MSC subpopulations have the potential to undergo neural differentiation, including marrow isolated adult multilineage inducible (MIAMI) cells, which differentiate into neuron-like cells in a multi-step neurotrophin 3-dependent manner. Epidermal and basic fibroblast growth factors are often used in neuronal differentiation protocols for MSCs, but with a limited understanding of their role. In this study, we thoroughly assessed for the first time the capacity of these factors to enhance the neuronal differentiation of MSCs. Materials and methods We have characterized MIAMI cell neuronal differentiation program in terms of stem cell molecule expression, cell cycle modifications, acquisition of a neuronal morphology and expression of neural and neuronal molecules in the absence and presence of an EGF-bFGF pre-treatment. Results EGF-bFGF pre-treatment down-regulated the expression of stemness markers Oct4A, Notch1 and Hes5, whereas neural/neuronal molecules Nestin, Pax6, Ngn2 and the neurotrophin receptor tyrosine kinase 1 and 3 were up-regulated. During differentiation, a sustained Erk phosphorylation in response to NT3 was observed, cells began to exit from the cell cycle and exhibit increased neurite-like extensions. In addition, neuronal β3-tubulin and neurofilament expression was increased; an effect mediated via the Erk pathway. A slight pre-oligodendrocyte engagement was noted, and no default neurotransmitter phenotype was observed. Overall, mesodermal markers were unaffected or decreased, while neurogenic/adipogenic PPARγ2 was incre ased. Conclusion EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells, further increasing their potential use in adult cell therapy of the nervous system.

Bone-marrow-derived stem cells — our key to longevity?

Bone marrow (BM) was for many years primarily regarded as the source of hematopoietic stem cells. In this review we discuss current views of the BM stem cell compartment and present data showing that BM contains not only hematopoietic but also heterogeneous non-hematopoietic stem cells. It is likely that similar or overlapping populations of primitive non-hematopoietic stem cells in BM were detected by different investigators using different experimental strategies and hence were assigned different names (e.g., mesenchymal stem cells, multipotent adult progenitor cells, or marrow-isolated adult multilineage inducible cells). However, the search still continues for true pluripotent stem cells in adult BM, which would fulfill the required criteria (e.g. complementation of blastocyst development). Recently our group has identified in BM a population of very small embryonic-like stem cells (VSELs), which express several markers characteristic for pluripotent stem cells and are found during early embryogenesis in the epiblast of the cylinder-stage embryo.

Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons

Marrow-isolated adult multilineage inducible (MIAMI) cells were differentiated in vitro to neuronal cells in a neurotrophin-dependent fashion. After induction, the cells revealed electrophysiological features similar to those observed in mature neurons. Primary early passage human MIAMI cells without any type of co-cultures with other cell types were used. The developmental program involved a multi-step process requiring the concerted action of brain-derived neurotrophic factor, nerve growth factor and depended on neurotrophin-3, after basic fibroblast growth factor withdrawal. MIAMI-derived neuron-like cells sequentially expressed the neuronal markers, developed a complex neurite outgrowth and arborization, and acquired electrophysiological characteristics similar to those observed in mature neurons. The young and old MIAMI-derived neuronal cells developed both inward and outward currents upon depolarization, similar to those observed in normal neurons. These results represent the earliest evidence that neurotrophin-3 can direct the differentiation of non-neural stem cells from human adult bone marrow stroma to neuron-like cells in vitro. Supplementing the aforementioned multi-step process with sonic hedgehog, fibroblast growth factor 8, and retinoic acid increased the expression of molecules involved in dopaminergic differentiation and of tyrosine hydroxylase, the rate limiting enzyme of dopamine synthesis. MIAMI cells from young and old individuals represent autologous human cell populations for the treatment of disorders of the skeletal and nervous systems and for applications in cell therapy and reparative medicine approaches.

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 (pO 2) in BM ranges from 1 to 7%, which prompted us to examine the role of pO 2 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-pO 2 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%) pO 2. Similarly, biomineralization of long-term cell cultures was high under osteoblastic differentiation conditions in air but was undetectable at low (3%) pO 2. In contrast, low pO 2 upregulated mRNAs for OCT-4, REX-1, telomerase reverse transcriptase, and hypoxia-inducible factor-1α, 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 pO 2 in the bone marrow, but tend to differentiate toward osteoblasts when they are located closer to blood vessels and exposed to higher pO 2. Our results strongly suggest that maintaining developmentally primitive human cells in vitro at low pO 2 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.

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.