Woosung Ahn

A new role of substance P as an injury-inducible messenger for mobilization of CD29 + stromal-like cells

Tissue injury may create a specific microenvironment for inducing the systemic participation of stromal-like cells in the repair process. Here we show that substance P is an injury-inducible factor that acts early in the wound healing process to induce CD29+ stromal-like cell mobilization. Likewise, mobilization of such cells also occurs in uninjured mice, rats and rabbits if substance P is intravenously injected. Upon further characterization these substance P–mobilized CD29+ cells were found to be similar to stromal cells from a number of connective tissues, including bone marrow (that is, bone marrow stromal cells, or BMSCs). Both substance P injection and transfusion of autologously derived substance P–mobilized CD29+ cells from uninjured rabbits accelerated wound healing in an alkali burn model. Also, epithelial engraftment of the transfused cells into the injured tissue occurred during the wound healing. Finally, using human BMSCs as a test population, we show that substance P stimulates transmigration, cell proliferation, activation of the extracellular signal–related kinases (Erk) 1 and 2 and nuclear translocation of β-catenin in vitro. This finding highlights a previously undescribed function of substance P as a systemically acting messenger of injury and a mobilizer of CD29+ stromal-like cells to participate in wound healing.

Substance P induces M2-type macrophages after spinal cord injury.

The potential benefits or the tissue-damaging effects of inflammatory response after central nervous system injuries have long been disputed. Recent studies have noted that substance P (SP), a neuropeptide, plays an important role in the wound-healing process by recruiting bone marrow stem cells to the injured tissue. In this study, we examined whether SP can enhance recovery from spinal cord injury (SCI) in Sprague–Dawley rats through its known function of stem cell mobilization and/or through the modulation of inflammation. We examined proinflammatory and anti-inflammatory cytokines and markers for macrophage subtypes. SP treatment modulated the SCI microenvironment toward a more anti-inflammatory and reparative one by inducing interleukin-10 and M2 macrophages and suppressing inducible nitric oxide synthase and tumor necrosis factor-&agr;. This modulation was achieved at 1 day much earlier than SP-stimulated bone marrow stem cells’ mobilization. Early intervention of the devastating inflammatory response by SP treatment caused the lesion cavity to become filled with robust axonal outgrowth that overlaid the M2 macrophages at 2 weeks – all of which culminated in tissue sparing and improvement in functional recovery from the SCI. SP is therefore a potential anti-inflammatory modulator for the treatment of injury-induced inflammatory central nervous system disorders.

Substance P reduces apoptotic cell death possibly by modulating the immune response at the early stage after spinal cord injury.

Previously, we have reported that substance P (SP) enhanced functional recovery from spinal cord injury (SCI) possibly by the anti-inflammatory modulation associated with the induction of M2-type macrophages at the injured lesion. In this study, we explored the cytokine expression profiles and apoptotic cell death in the lesion site of the SCI after an immediate intravenous injection of SP. SP injection increased the levels of interleukin-4 (IL-4), IL-6, and IL-10 at day 1 after the SCI approximately by 2-, 9-, and 10-folds when compared with the control SCI, respectively. On the basis of double immunofluorescence staining with IL-10 and CD11b, activated macrophages or microglia expressing IL-10 appeared in the margin of the lesion site at day 1 only after the SP injection. This SP-mediated alteration in the lesion microenvironment was shown to be associated with the lower cell death of neuronal cells at day 1 and oligodendrocytes at day 5 by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which was also accompanied by a decrease in caspase-3 activation. These findings suggest that SP may reduce the inflammation-induced secondary cell death, possibly through immune modulation at an early stage after the SCI.

Induction of mesenchymal to epithelial transition of circulating mesenchymal stem cells by conditioned medium of injured cornea

We previously reported that substance-P (SP) mobilized mesenchymal stem cells (MSCSP) from bone marrow and recruited them to the injured site, which then participated in wound healing process by incorporation into the corneal epithelium in a rabbit alkali burn model. In the present study, we investigated if the injured microenvironment could stimulate the recruited MSCSP to lose their mesenchymal phenotype and then acquire epithelial phenotype or not. Especially, we focused on the effects of secreted soluble tissue factors of wound microenvironment in the rat cornea wound model. Priming the circulating MSCSP with conditioned medium of the injured tissue at day 5 elicited morphological and molecular changes similar to those noted during mesenchymal to epithelial transition (MET), which was accompanied by the reduction of alpha-smooth muscle actin (α-SMA), a typical mesenchymal marker, and induction of E-cadherin, a typical epithelial marker. Furthermore, these primed MSCSP rearranged their actin cytoskeletal system from distinct actin stress fiber projection to the cell-to-cell-contact-enriched actin microfilament. The primed cells also had increased survival in keratinocyte growth medium (KGM). In conclusion, MSCSP could be differentiated in vitro to the epithelial cells under the injured microenvironment, which may also occur in vivo during tissue repair.

CXCL5 abundant in the wound fluid at the late phase of wound healing, possibly promoting migration of mesenchymal stem cells and vascular tube formation

We previously reported that wound fluid generated from injured cornea at 5 day after injury had ability to induce mesenchymal to epithelial transition (MET) of circulating mesenchymal stem cells (MSCs). In the present study, we further analyzed wound fluid by cytokine array. Comparing with normal cornea, increased expression of chemokines CXCL2, CXCL3, CXCL5, CCL2, anti-inflammatory cytokine IL-10, growth factors VEGF and β-NGF, protease inhibitor TIMP-1 were observed in the wound fluid of injured cornea. Interestingly, the inductions of inflammatory cytokines, IL-1α, IL-1β, IL-4, IL-6, and TNFα were not detected. It suggests wound fluid at 5 day after injury consisted of beneficial mediators for wound healing. In particular, chemokine CXCL5 increased over 6 fold. We further analyzed the roles of CXCL5 using MSCs, isolated from bone marrow and human umbilical vein endothelial cells (HUVECs). Administration of CXCL5 significantly enhanced motility of MSCs as well as showed chemotactic effects on MSCs. Moreover, CXCL5 increased the endothelial proliferation and vascular density and spouting determined by vessel formation assay. Taken together, wound fluids at day 5 after injury are the pool of beneficial mediators for wound healing process.

Identifying a molecular and cellular phenotype of mesenchymal stem cells mobilized from substance P in the peripheral blood

Here we compared MSCs isolated from bone marrow (BMSC) and peripheral blood at 2 days after intravenous substance P (SP) injection (MSCSP) to define specific roles of circulating MSCs in tissue repair process. The mass analysis of changed genes over and under log2 fold showed 97% of genes were identical in these two groups. We found MSCSP increased the molecular signatures related to homing and engraftment (tetraspanin 8, tetraspanin 12), extracellular matrix (ECM) (collagen type IV α1, nidogen 2, laminin-α5, basement membrane components; collagen type XII α1, collagen XV α1, ECM components linking basement membrane to stroma), transmigration (ICAM-1, P-selectin, endothelin), motility (Rho GTPase activating protein 8, myosin heavy chain 10 and 11, troponin C type 1 and 2), transdifferentiation potential (keratin 19, CMTM 8), immunity (IL-1α, IL-33, IL-1R type 1), and angiogenesis (endothelin1, VEGF-C, IL-33, laminin-α5) according to functional classification of the 3 % of changed genes. Functional differences were also shown in MSCSP in regulation of viability of immune cells, Jurkat T cells and U937 monocytes. In addition, a-smooth muscle actin (α-SMA) significantly increased in MSCSP compared with BMSC, demonstrating committed differentiation to myofibroblast participated in tissue repair. However, intrinsic differentiation potential to mesenchymal tissues, adipogenesis and osteogenesis, was not altered in MSCSP. From the molecular and cellular phenotyping of MSCSP, circulating MSCSP seems to be a distinct population mobilized from heterogenous BMSC with well adapted for the participation in tissue repair process by enhancing homing, engraftment, migration, and angiogenesis with multipotent differentiation potential to either myofibroblasts or epithelial-like cells.

CD45+/CD11b+ monocytes are required for mesenchymal stem cell proliferation In Vitro

Mesenchymal stem cells (MSCs) have been used to treat patients with incurable diseases. However, the low yield of MSCs even after long periods of ex vivo culture remains a serious problem. Several attempts were made to increase cell expansion rate of MSCs in cultures. In this study, we examined the stimulatory effect of monocytes on the proliferation and colony forming efficiency of MSCs by co-culturing CD29+/CD45-MSCs with CD45+/ CD11b+ monocytes. In presence of CD45+/CD11b+ monocytes, CD29+/CD45-MSCs proliferated rapidly to form large and compact colonies within 10 days. In the absence of CD45+/CD11b+ monocytes, CD29+/CD45-MSC colony formation was much delayed, indicating that the presence of CD45+/CD11b+ monocytes is required for CD29+/ CD45-MSC proliferation and stemness in vitro. Similar growth-stimulating effect on MSC culture was also found when conditioned medium from CD45+/CD11b+ monocyte cultures was supplied instead of CD45+/CD11b+ monocytes. These results suggest that secretory molecules of CD45+/CD11b+ monocytes may mainly mediate the enhanced growth of CD29+/CD45-MSCs. Collectively, this study reveals that co-culture of CD29+/CD45-MSCs with CD45+/CD11b+ monocytes stimulate MSC growth via paracrine factors, which could improve colony forming capacity of MSC. Furthermore, conditioned medium of CD45+/CD11b+ monocytes may also be developed for cell culture supplement for MSC cell therapeutics.

Endothelial precursor cells stimulate pericyte-like coverage of bone marrow-derived mesenchymal stem cells through platelet-derived growth factor-BB induction, which is enhanced by substance P

The aim of this study was to evaluate the angiogenicity of a combination of BM‐EPCs and BM‐MSCs in vitro in the presence of SP and its working mechanism.

Comparative Analysis of the Cell Fates of Induced Schwann Cells from Subcutaneous Fat Tissue and Naïve Schwann Cells in the Sciatic Nerve Injury Model

Purpose The fate and function of the induced Schwann cells (iSCs) like cells from adipose tissue have not been critically evaluated in vivo after transplantation. The objective of this study is to compare the fate of iSCs with naïve SCs (nSCs) after transplantation into the lesion sites of sciatic nerve, respectively. Methods Adipose-derived stem cells from eGFP-expressing transgenic rats subcutaneous fat were induced to iSCs in vitro. iSCs were injected to the sciatic nerve lesion area after crush injury and the cells fate was comparatively analyzed with that of nSCs from the same rat. Results At 12 weeks after transplantation, nSCs were detected only in the restricted area of cell transplantation site but iSCs were widely distributed all over the sciatic nerve. Based on double fluorescence observations, both iSCs and naïve ones were colocalized with P0-expressing myelin sheath, outbound by laminin-expressing basal membrane, and terminated at contactin-associated protein-expressing doublets. However, some of iSCs were also differentiated to the fibrocyte/fibroblast-like cells. In the histological analysis of repaired sciatic nerves, axon density was higher in iSC-received group than in the nSCs group and normal sciatic nerve. Conclusion iSCs induced from subcutaneous fat tissues have higher engraftment and migration capacity than nSCs.