Jeffrey L. Spees

Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice

We tested the hypothesis that multipotent stromal cells from human bone marrow (hMSCs) can provide a potential therapy for human diabetes mellitus. Severe but nonlethal hyperglycemia was produced in NOD/scid mice with daily low doses of streptozotocin on days 1–4, and hMSCs were delivered via intracardiac infusion on days 10 and 17. The hMSCs lowered blood glucose levels in the diabetic mice on day 32 relative to untreated controls (18.34 mM ± 1.12 SE vs. 27.78 mM ± 2.45 SE, P = 0.0019). ELISAs demonstrated that blood levels of mouse insulin were higher in the hMSC-treated as compared with untreated diabetic mice, but human insulin was not detected. PCR assays detected human Alu sequences in DNA in pancreas and kidney on day 17 or 32 but not in other tissues, except heart, into which the cells were infused. In the hMSC-treated diabetic mice, there was an increase in pancreatic islets and β cells producing mouse insulin. Rare islets contained human cells that colabeled for human insulin or PDX-1. Most of the β cells in the islets were mouse cells that expressed mouse insulin. In kidneys of hMSC-treated diabetic mice, human cells were found in the glomeruli. There was a decrease in mesangial thickening and a decrease in macrophage infiltration. A few of the human cells appeared to differentiate into glomerular endothelial cells. Therefore, the results raised the possibility that hMSCs may be useful in enhancing insulin secretion and perhaps improving the renal lesions that develop in patients with diabetes mellitus.

Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma.

To investigate stem cell differentiation in response to tissue injury, human mesenchymal stem cells (hMSCs) were cocultured with heat-shocked small airway epithelial cells. A subset of the hMSCs rapidly differentiated into epithelium-like cells, and they restored the epithelial monolayer. Immunocytochemistry and microarray analyses demonstrated that the cells expressed many genes characteristic of normal small airway epithelial cells. Some hMSCs differentiated directly after incorporation into the epithelial monolayer but other hMSCs fused with epithelial cells. Surprisingly, cell fusion was a frequent rather than rare event, in that up to 1% of the hMSCs added to the coculture system were recovered as binucleated cells expressing an epithelial surface epitope. Some of the fused cells also underwent nuclear fusion.

Mitochondrial transfer between cells can rescue aerobic respiration

Current theory indicates that mitochondria were obtained 1.5 billion years ago from an ancient prokaryote. The mitochondria provided the capacity for aerobic respiration, the creation of the eukaryotic cell, and eventually complex multicellular organisms. Recent reports have found that mitochondria play essential roles in aging and determining lifespan. A variety of heritable and acquired diseases are linked to mitochondrial dysfunction. We report here that mitochondria are more dynamic than previously considered: mitochondria or mtDNA can move between cells. The active transfer from adult stem cells and somatic cells can rescue aerobic respiration in mammalian cells with nonfunctional mitochondria.

Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo.

The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1α in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.

How Wnt Signaling Affects Bone Repair by Mesenchymal Stem Cells from the Bone Marrow

Abstract: Human mesenchymal stem cells (hMSCs) from bone marrow are a source of osteoblast progenitors in vivo, and under appropriate conditions they differentiate into osteoblasts ex vivo. The cells provide a convenient cell culture model for the study of osteogenic tissue repair in an experimentally accessible system. Recent advances in the field of skeletal development and osteogenesis have demonstrated that signaling through the canonical wingless (Wnt) pathway is critical for the differentiation of progenitor cell lines into osteoblasts. Inhibition of such signals can predispose hMSCs to cell cycle entry and prevent osteogenesis. Our investigation of the role of Wnt signaling in osteogenesis by hMSCs ex vivo has demonstrated that osteogenesis proceeds in response to bone morphogenic protein 2 stimulation and is sustained by Wnt signaling. In the presence of Dkk‐1, an inhibitor of Wnt signaling, the cascade is disrupted, resulting in inhibition of osteogenesis. Peptide mapping studies have provided peptide Dkk‐1 agonists and the opportunity for the production of blocking antibodies. Anti‐Dkk‐1 strategies are clinically relevant since high serum levels of Dkk‐1 are thought to contribute to osteolytic lesion formation in multiple myeloma and possibly some forms of osteosarcoma. Specific inhibitors of glycogen synthetase kinase 3β (GSK3β), which mimic Wnt signaling, may also have a therapeutic benefit by enhancing in vitro osteogenesis despite the presence of Dkk‐1. Antibodies that block Dkk‐1 and GSK3β inhibitors may provide novel opportunities for the enhancement of bone repair in a variety of human diseases such as multiple myeloma and osteosarcoma.

Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension

Infusion of bone marrow stem or progenitor cells may provide powerful therapies for injured tissues such as the lung and heart. We examined the potential of bone marrow‐derived (BMD) progenitor cells to contribute to repair and remodeling of lung and heart in a rat monocrotaline (MCT) model of pulmonary hypertension. Bone marrow from green fluorescent protein (GFP)‐transgenic male rats was transplanted into GFP‐negative female rats. The chi‐meric animals were injected with MCT to produce pulmonary hypertension. Significant numbers of male GFP‐positive BMD cells engrafted in the lungs of MCT‐treated rats. Microarray analyses and double‐im‐munohistochemistry demonstrated that many of the cells were interstitial fibroblasts or myofibroblasts, some of the cells were hematopoietic cells, and some were pulmonary epithelial cells (Clara cells), vascular endothelial cells, and smooth muscle cells. A few BMD cells fused with pulmonary cells from the host, but the frequency was low. In the hypertrophied hearts of MCT‐treated rats, we found a significant increase in the relative numbers of BMD cells in the right ventricle wall as compared with the left ventricle. Some of the BMD cells in the right ventricle were vascular cells and cardiomyocytes. We report BMD cardiomyocytes with a normal chromosome number, fusion of BMD cells with host cardiomyocytes, and, in some cases, nuclear fusion. Spees, J. L., Whitney, M. J., Sullivan, D. E., Lasky, J. A., Laboy, M., Ylostalo, J., Prockop, D. J. Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension. FASEB J. 22, 1226–1236 (2008)

Mechanisms of mesenchymal stem/stromal cell function

The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell–cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics.

CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.

Transplantation of Nonhematopoietic Adult Bone Marrow Stem/Progenitor Cells Isolated by p75 Nerve Growth Factor Receptor Into the Penis Rescues Erectile Function in a Rat Model of Cavernous Nerve Injury

PURPOSE Radical prostatectomy for prostate cancer frequently results in erectile dysfunction and decreased quality of life. We investigated the effects of transplanting nonhematopoietic adult bone marrow stem/progenitor cells (multipotent stromal cells) into the corpus cavernosum in a rat model of bilateral cavernous nerve crush injury. MATERIALS AND METHODS Multipotent stromal cells were isolated from the bone marrow of transgenic green fluorescent protein rats by plastic adherence (rat multipotent stromal cells) or magnetic activated cell sorting using antibodies against p75 low affinity nerve growth factor receptor (p75 derived multipotent stromal cells). Bilateral cavernous nerve crush injury was induced in adult male Sprague-Dawley rats. Immediately after injury 8 rats each were injected intracavernously with phosphate buffered saline (vehicle control), fibroblasts (cell control), rat multipotent stromal cells (cell treatment) or p75 derived multipotent stromal cells (cell treatment). Another 8 rats underwent sham operation (phosphate buffered saline injection). Four weeks after the procedures we assessed erectile function by measuring the intracavernous-to-mean arterial pressure ratio and total intracavernous pressure during cavernous nerve stimulation. RESULTS Intracavernous injection of p75 derived multipotent stromal cells after bilateral cavernous nerve crush injury resulted in a significantly higher mean intracavernous-to-mean arterial pressure ratio and total intracavernous pressure compared with all other groups except the sham operated group (p <0.05). Rats injected with typical multipotent stromal cells had partial erectile function rescue compared with animals that received p75 derived multipotent stromal cells. Fibroblast (cell control) and phosphate buffered saline (vehicle control) injection did not improve erectile function. Enzyme-linked immunosorbent assay suggested that basic fibroblast growth factor secreted by p75 derived multipotent stromal cells protected the cavernous nerve after bilateral cavernous nerve crush injury. CONCLUSIONS Transplantation of adult stem/progenitor cells may provide an effective treatment for erectile dysfunction after radical prostatectomy.

Isolation of Locally Derived Stem/Progenitor Cells From the Peri-Infarct Area That Do Not Migrate From the Lateral Ventricle After Cortical Stroke

Background and Purpose— Neurogenesis can arise from neural stem/progenitor cells of the subventricular zone after strokes involving both the cortex and striatum. However, it is controversial whether all types of stroke and strokes of different sizes activate neurogenesis from the subventricular zone niche. In contrast with cortical/striatal strokes, repair and remodeling after mild cortical strokes may involve to a greater extent local cortical stem/progenitor cells and cells from nonneurogenic niches. Methods— We compared stem/progenitor cell responses after focal cortical strokes produced by distal middle cerebral artery occlusion and cortical/striatal strokes produced by the intraluminal suture model. To label migrating neuroblasts from the subventricular zone, we injected DiI to the lateral ventricle after distal middle cerebral artery occlusion. By immunohistochemistry, we characterized cells expressing stem/progenitor cell markers in the peri-infarct area. We isolated cortical stem/progenitor cells from the peri-infarct area after distal middle cerebral artery occlusion and assayed their self-renewal and differentiation capacity. Results— In contrast with cortical/striatal strokes, focal cortical strokes did not induce neuroblast migration from the subventricular zone to the infarct zone after distal middle cerebral artery occlusion. By immunohistochemistry, we observed subpopulations of reactive astrocytes in the peri-infarct area that coexpressed radial glial cell markers such as Sox2, Nestin, and RC2. Clonal neural spheres isolated from the peri-infarct area after distal middle cerebral artery occlusion differentiated into neurons, astrocytes, oligodendrocytes, and smooth muscle cells. Notably, neural spheres isolated from the peri-infarct area also expressed RC2 before differentiation. Conclusions— Mild cortical strokes that do not penetrate the striatum activate local cortical stem/progenitor cells but do not induce neuroblast migration from the subventricular zone niche.