SUMMARY Quantitative assays for human DNA and mRNA were used to examine the paradox that intravenously (i.v.) infused human multipotent stromal cells (hMSCs) can enhance tissue repair without significant engraftment. After 2 × 106 hMSCs were i.v. infused into mice, most of the cells were trapped as emboli in lung. The cells in lung disappeared with a half-life of about 24 hr, but <1000 cells appeared in six other tissues. The hMSCs in lung upregulated expression of multiple genes, with a large increase in the anti-inflammatory protein TSG-6. After myocardial infarction, i.v. hMSCs, but not hMSCs transduced with TSG-6 siRNA, decreased inflammatory responses, reduced infarct size, and improved cardiac function. I.v. administration of recombinant TSG-6 also reduced inflammatory responses and reduced infarct size. The results suggest that improvements in animal models and patients after i.v. infusions of MSCs are at least in part explained by activation of MSCs to secrete TSG-6.

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 NOD͞scid 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 ␤ cells producing mouse insulin. Rare islets contained human cells that colabeled for human insulin or PDX-1. Most of the ␤ 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.insulin ͉ pancreas ͉ streptozotocin ͉ transplantation P revious publications presented conflicting observations as to whether cells from bone marrow can provide a potential therapy for diabetes mellitus. One strategy (1-4) was to differentiate plastic adherent marrow cells in culture into insulin-secreting cells. A second strategy was to transplant diabetic mice with genetically labeled marrow and to search for labeled insulinproducing cells in the recipient mice. One study using a CRELoxP-GFP system found that 1.7-3% of the cells in islets of the recipient mice were marrow-derived and that GFP-labeled donor cells isolated from the islets expressed insulin, glucose transporter 2, and transcription factors typically found in ␤ cells (5). Three subsequent reports in which mice were transplanted with GFPexpressing bone marrow did not find evidence of marrow cells becoming insulin-producing cells in the pancreas of recipient mice (6-8), but in the reports it was difficult to exclude the possibility that the GFP gene was inactivated or that GFP-labeled cells were destroyed as they engrafted into islets. A third strategy was to determine whether systemically administered marrow cells enhanced regeneration of pancreatic insulin-producing cells in diabetic models. Hess et al. (9) reported that in NOD͞scid mice in which diabetes was induced with streptozotocin (STZ), partial marrow ablation followed by transplantation of either GFP-labeled whole-marrow or GFP-labeled c-kit ϩ cells from murine marrowenhanced r...

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We screened for surface proteins expressed only by the early progenitor cells present in low-passage, low-density cultures of the adult stem/progenitor cells from bone marrow referred to as mesenchymal stem cells or multipotent stromal cells (MSCs). Six proteins were identified that were selectively expressed in the early progenitors: podocalyxin-like protein (PODXL), ␣6-integrin (CD49f), ␣4-integrin (CD49d), c-Met, CXCR4, and CX3CR1. All were previously shown to be involved in cell trafficking or tumor progression. Antibodies to CD49f and PODXL, a sialomucin in the CD34 family, were the most robust for FACScan assays. PODXL hi /CD49f hi MSCs were more clonogenic and differentiated more efficiently than PODXL lo /CD49f lo cells. Inhibition of expression of PODXL with RNA interference caused aggregation of the cells. Furthermore, PODXL hi /CD49f hi MSCs were less prone to produce lethal pulmonary emboli, and larger numbers were recovered in heart and kidney after intravenous infusion into mice with myocardial infarcts. (Blood. 2009;113:816-826) IntroductionAmong the cells being used for cell therapies for nonhematopoietic tissues are the stem/progenitor cells from bone marrow that were referred to initially as fibroblast colony-forming units, 1 subsequently as marrow stromal cells, then as mesenchymal stem cells 2 and, most recently, as multipotent mesenchymal stromal cells or MSCs. 3 Clinical trials with MSCs are now in progress, 4-9 but several questions are still unresolved as to how the cells should be isolated, expanded in culture, and characterized. One view is that confluent cultures of MSCs ( Figure 1A) are useful and perhaps the optimal preparations for therapy. An opposing view is that confluent cultures of MSCs are partially committed to differentiation or even senescence. Therefore, they lack some of the therapeutic potentials of low-density cultures that contain a subpopulation of rapidly self-replicating cells [10][11][12][13] that display a different pattern of expressed genes, 14 that have a greater capacity to generate singlecell-derived clones, 10,11 and that more efficiently engraft in vivo. 15 MSCs were originally described by Friedenstein et al 1 and Owen and Friedenstein,16 who isolated the cells by their ready adherence to tissue culture surfaces, an isolation technique that subsequently was followed by most investigators. 17 The cells were characterized primarily by their ability to generate colonies in culture and to differentiate into adipocytes, osteoblasts, and chondrocytes. Numerous attempts were made to develop more specific procedures for isolation and characterization of the cells by preparing antibodies to surface epitopes on MSC. The first antibody was the monoclonal immunoglobulin M antibody STRO-1, which was raised against confluent cultures of human MSCs that were used as feeder layers for hematopoietic stem cells. 18 STRO-1 alone or in combination with other antibodies subsequently was used extensively to identify and isolate MSCs. [19][20][21][22][23][24][25][26] Also, a se...

Stromal-derived factor-1 (SDF-1)-mediated CXCR4 signaling plays important roles in migration, engraftment, and proliferation of stem cells. We report here that CXCR4 overexpression on human adipose tissue stromal cells (hADSCs) using a lentiviral gene transfer technique helped navigate these cells to the injured tissues in response to SDF-1 signaling. Transduced hADSCs, expressing high levels of CXCR4, displayed an increased capacity for cellular growth and protection against etoposide-induced cell death. CXCR4-overexpressed cells showed higher ERK activity than that of vector-transduced cells. U0126, an ERK inhibitor, and AMD3100, a CXCR4 antagonist, inhibited the proliferation of CXCR4 overexpression-induced proliferation and ERK phosphorylation. CXCR4-overexpressing cells showed increased level of beta-catenin and luciferase activity driven by the Tcf promoter. Our results suggest CXCR4 overexpression for improved hADSC motility, retention, and proliferation could be beneficial for in vivo navigation and expansion of stem cells.