Ryang Hwa Lee

Intravenous hMSCs Improve Myocardial Infarction in Mice because Cells Embolized in Lung Are Activated to Secrete the Anti-inflammatory Protein TSG-6

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 x 10(6) 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.

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.

Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-κB signaling in resident macrophages.

Human mesenchymal stem/progenitor cells (hMSCs) repair tissues and modulate immune systems but the mechanisms are not fully understood. We demonstrated that hMSCs are activated by inflammatory signals to secrete the anti-inflammatory protein, TNF-α-stimulated gene 6 protein (TSG-6) and thereby create a negative feedback loop that reduces inflammation in zymosan-induced peritonitis. The results demonstrate for the first time that TSG-6 interacts through the CD44 receptor on resident macrophages to decrease zymosan/TLR2-mediated nuclear translocation of the NF-κB. The negative feedback loop created by MSCs through TSG-6 attenuates the inflammatory cascade that is initiated by resident macrophages and then amplified by mesothelial cells and probably other cells of the peritoneum. Because inflammation underlies many pathologic processes, including immune responses, the results may explain the beneficial effects of MSCs and TSG-6 in several disease models.

Action at a Distance: Systemically Administered Adult Stem/Progenitor Cells (MSCs) Reduce Inflammatory Damage to the Cornea Without Engraftment and Primarily by Secretion of TNF‐α Stimulated Gene/Protein 6

Previous reports demonstrated that the deleterious effects of chemical injury to the cornea were ameliorated by local or systemic administration of adult stem/progenitor cells from bone marrow referred to as mesenchymal stem or stromal cells (MSCs). However, the mechanisms for the beneficial effects of MSCs on the injured cornea were not clarified. Herein, we demonstrated that human MSCs (hMSCs) were effective in reducing corneal opacity and inflammation without engraftment after either intraperitoneal (i.p.) or intravenous (i.v.) administration following chemical injury to the rat cornea. A quantitative assay for human mRNA for glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) demonstrated that less than 10 hMSCs were present in the corneas of rats 1‐day and 3 days after i.v. or i.p. administration of 1 × 107 hMSCs. In vitro experiments using a transwell coculture system demonstrated that chemical injury to corneal epithelial cells activated hMSCs to secrete the multipotent anti‐inflammatory protein TNF‐α stimulated gene/protein 6 (TSG‐6). In vivo, the effects of i.v. injection of hMSCs were largely abrogated by knockdown of TSG‐6. Also, the effects of hMSCs were essentially duplicated by either i.v. or topical administration of TSG‐6. Therefore, the results demonstrated that systemically administered hMSCs reduce inflammatory damage to the cornea without engraftment and primarily by secretion of the anti‐inflammatory protein TSG‐6 in response to injury signals from the cornea. STEM CELLS 2011;29:1572–1579

The CD34-like protein PODXL and α6-integrin (CD49f) identify early progenitor MSCs with increased clonogenicity and migration to infarcted heart in mice

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), alpha6-integrin (CD49f), alpha4-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.

Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α–stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.

Intravenous Mesenchymal Stem Cells Prevented Rejection of Allogeneic Corneal Transplants by Aborting the Early Inflammatory Response

Mesenchymal stem/progenitor cells (MSCs) were reported to enhance the survival of cellular and organ transplants. However, their mode of action was not established. We here used a mouse model of corneal allotransplantation and demonstrated that peri-transplant intravenous (i.v.) infusion of human MSCs (hMSCs) decreased the early surgically induced inflammation and reduced the activation of antigen-presenting cells (APCs) in the cornea and draining lymph nodes (DLNs). Subsequently, immune rejection was decreased, and allograft survival was prolonged. Quantitative assays for human GAPDH revealed that <10 hMSCs out of 1 × 10(6) injected cells were recovered in the cornea 10 hours to 28 days after i.v. infusion. Most of hMSCs were trapped in lungs where they were activated to increase expression of the gene for a multifunctional anti-inflammatory protein tumor necrosis factor-α stimulated gene/protein 6 (TSG-6). i.v. hMSCs with a knockdown of TSG-6 did not suppress the early inflammation and failed to prolong the allograft survival. Also, i.v. infusion of recombinant TSG-6 reproduced the effects of hMSCs. Results suggest that hMSCs improve the survival of corneal allografts without engraftment and primarily by secreting TSG-6 that acts by aborting early inflammatory responses. The same mechanism may explain previous reports that MSCs decrease rejection of other organ transplants.

Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that enhances expression of type II collagen

OBJECTIVE Meniscal regeneration was previously shown to be enhanced by injection of mesenchymal stem/stromal cells (MSCs) but the mode of action of the MSCs was not established. The aim of this study was to define how injection of MSCs enhances meniscal regeneration. DESIGN A hemi-meniscectomy model in rats was used. Rat-MSCs (rMSCs) or human-MSCs (hMSCs) were injected into the right knee joint after the surgery, and PBS was injected into the left. The groups were compared macroscopically and histologically at 2, 4, and 8 weeks. The changes in transcription in both human and rat genes were assayed by species-specific microarrays and real-time RT-PCRs. RESULTS Although the number of hMSCs decreased with time, hMSCs enhanced meniscal regeneration in a manner similar to rMSCs. hMSCs injection increased expression of rat type II collagen (rat-Col II), and inhibited osteoarthritis progression. The small fraction of hMSCs was activated to express high levels of a series of genes including Indian hedgehog (Ihh), parathyroid hormone-like hormone (PTHLH), and bone morphogenetic protein 2 (BMP2). The presence of hMSCs triggered the subsequent expression of rat-Col II. An antagonist of hedgehog signaling inhibited the expression of rat-Col II and an agonist increased expression of rat-Col II in the absence of hMSCs. CONCLUSIONS Despite rapid reduction in cell numbers, intra-articular injected hMSCs were activated to express Ihh, PTHLH, and BMP2 and contributed to meniscal regeneration. The hedgehog signaling was essential in enhancing the expression of rat-Col II, but several other factors provided by the hMSCs probably contributed to the repair.

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.

TSG-6 as a biomarker to predict efficacy of human mesenchymal stem/progenitor cells (hMSCs) in modulating sterile inflammation in vivo

Significance The clinical trials with human mesenchymal stem/progenitor cells (hMSCs) from bone marrow and other tissues are proceeding even though cultures of the cells are heterogeneous and there is large variability among preparations of hMSCs due to differences among donors, culture conditions, and inconsistent tissue sampling. However, there is currently no in vitro bioassay for the evaluation of hMSC efficacy in vivo. Therefore, the value of the data obtained from current clinical trials may well be compromised by variations in the quality of the hMSCs used. This study provides, to our knowledge, the first biomarker that can predict the efficacy of hMSCs in suppressing sterile inflammation in vivo. Human mesenchymal stem/progenitor cells (hMSCs) from bone marrow and other tissues are currently being administered to large numbers of patients even though there are no biomarkers that accurately predict their efficacy in vivo. Using a mouse model of chemical injury of the cornea, we found that bone-marrow–derived hMSCs isolated from different donors varied widely in their efficacy in modulating sterile inflammation. Importantly, RT-PCR assays of hMSCs for the inflammation-modulating protein TSG-6 expressed by the TNFα-stimulated gene 6 (TSG-6 or TNFAIP6) predicted their efficacy in sterile inflammation models for corneal injury, sterile peritonitis, and bleomycin-induced lung injury. In contrast, the levels of TSG-6 mRNA were negatively correlated with their potential for osteogenic differentiation in vitro and poorly correlated with other criteria for evaluating hMSCs. Also, a survey of a small cohort suggested that hMSCs from female donors compared with male donors more effectively suppressed sterile inflammation, expressed higher levels of TSG-6, and had slightly less osteogenic potential.