Insung Kang

A p38 MAPK-Mediated Alteration of COX-2/PGE2 Regulates Immunomodulatory Properties in Human Mesenchymal Stem Cell Aging

Because human mesenchymal stem cells (hMSC) have profound immunomodulatory effects, many attempts have been made to use hMSCs in preclinical and clinical trials. For hMSCs to be used in therapy, a large population of hMSCs must be generated by in vitro expansion. However, the immunomodulatory changes following the in vitro expansion of hMSCs have not been elucidated. In this study, we evaluated the effect of replicative senescence on the immunomodulatory ability of hMSCs in vitro and in vivo. Late-passage hMSCs showed impaired suppressive effect on mitogen-induced mononuclear cell proliferation. Strikingly, late-passage hMSCs had a significantly compromised protective effect against mouse experimental colitis, which was confirmed by gross and histologic examination. Among the anti-inflammatory cytokines, the production of prostaglandin E2 (PGE2) and the expression of its primary enzyme, cyclooxygenase-2 (COX-2), were profoundly increased by pre-stimulation with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and this response was significantly decreased with consecutive passages. We demonstrated that the impaired phosphorylation activity of p38 MAP kinase (p38 MAPK) in late-passage hMSCs led to a compromised immunomodulatory ability through the regulation of COX-2. In conclusion, our data indicate that the immunomodulatory ability of hMSCs gradually declines with consecutive passages via a p38-mediated alteration of COX-2 and PGE2 levels.

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.

Inducible HGF-secreting Human Umbilical Cord Blood-derived MSCs Produced via TALEN-mediated Genome Editing Promoted Angiogenesis

Mesenchymal stem cells (MSCs) promote therapeutic angiogenesis to cure serious vascular disorders. However, their survival period and cytokine-secretory capacity are limited. Although hepatocyte growth factor (HGF) can accelerate the rate of angiogenesis, recombinant HGF is limited because of its very short half-life (<3–5 minutes). Thus, continuous treatment with HGF is required to obtain an effective therapeutic response. To overcome these limitations, we produced genome-edited MSCs that secreted HGF upon drug-specific induction. The inducible HGF expression cassette was integrated into a safe harbor site in an MSC chromosome using the TALEN system, resulting in the production of TetOn-HGF/human umbilical cord blood-derived (hUCB)-MSCs. Functional assessment of the TetOn-HGF/hUCB-MSCs showed that they had enhanced mobility upon the induction of HGF expression. Moreover, long-term exposure by doxycycline (Dox)-treated TetOn-HGF/hUCB-MSCs enhanced the anti-apoptotic responses of genome-edited MSCs subjected to oxidative stress and improved the tube-formation ability. Furthermore, TetOn-HGF/hUCB-MSCs encapsulated by arginine-glycine-aspartic acid (RGD)-alginate microgel induced to express HGF improved in vivo angiogenesis in a mouse hindlimb ischemia model. This study showed that the inducible HGF-expressing hUCB-MSCs are competent to continuously express and secrete HGF in a controlled manner. Thus, the MSCs that express HGF in an inducible manner are a useful therapeutic modality for the treatment of vascular diseases requiring angiogenesis.

miR-410 Inhibition Induces RPE Differentiation of Amniotic Epithelial Stem Cells via Overexpression of OTX2 and RPE65

The retinal pigment epithelium (RPE) is a highly specialized cell type located between the choroid and neural retina of the eye. RPE degeneration causes irreversible visual impairment, extending to blindness. Cell therapy has recently emerged as a potential therapeutic approach for retinal degeneration. MicroRNA-based differentiation of stem cells is a new strategy for producing tissue-specific cell types. In this study, we developed a novel microRNA-based strategy for RPE induction from human amniotic epithelial stem cells (AESCs). We identified microRNAs involved in RPE development in AESCs. Of 29 putative human RPE-relevant microRNAs, microRNA-410 (miR-410) was predicted to target multiple RPE development-relevant genes. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including OTX2, RPE65, Bestrophin and EMMPRIN. These RPE-like cells were morphologically altered toward a cobblestone-like shape and were able to phagocytize microbeads. We showed that miR-410 directly regulates predicted target genes OTX2 and RPE65. Our microRNA-based strategy demonstrated RPE differentiation in AESCs by treatment of an antisense microRNA-410 (anti-miR-410), without the use of additional factors or exogenous transduction. These findings suggest that miR-410 inhibition can be a useful tool for directed cell differentiation and an attractive method for cell therapy in human retinal degenerative diseases.

Excessive microglial activation aggravates olfactory dysfunction by impeding the survival of newborn neurons in the olfactory bulb of Niemann–Pick disease type C1 mice

Progressive olfactory impairment is one of the earliest markers of neurodegeneration. However, the underlying mechanism for this dysfunction remains unclear. The present study investigated the possible role of microgliosis in olfactory deficits using a mouse model of Niemann-Pick disease type C1 (NPC1), which is an incurable neurodegenerative disorder with disrupted lipid trafficking. At 7weeks of age, NPC1 mutants showed a distinct olfactory impairment in an olfactory test compared with age-matched wild-type controls (WT). The marked loss of olfactory sensory neurons within the NPC1 affected olfactory bulb (NPC1-OB) suggests that NPC1 dysfunction impairs olfactory structure. Furthermore, the pool of neuroblasts in the OB was diminished in NPC1 mice despite the intact proliferative capacity of neural stem/progenitor cells in the subventricular zone. Instead, pro-inflammatory proliferating microglia accumulated extensively in the NPC1-OB as the disease progressed. To evaluate the impact of abnormal microglial activation on olfaction in NPC1 mice, a microglial inhibition study was performed using the anti-inflammatory agent Cyclosporin A (CsA). Importantly, long-term CsA treatment in NPC1 mice reduced reactive microgliosis, restored the survival of newly generated neurons in the OB and improved overall performance on the olfactory test. Therefore, our study highlights the possible role of microglia in the regulation of neuronal turnover in the OB and provides insight into the possible therapeutic applications of microglial inhibition in the attenuation or reversal of olfactory impairment.

Cathepsin S contributes to microglia-mediated olfactory dysfunction through the regulation of Cx3cl1–Cx3cr1 axis in a Niemann–Pick disease type C1 model

Microglia can aggravate olfactory dysfunction by mediating neuronal death in the olfactory bulb (OB) of a murine model of Niemann–Pick disease type C1 (NPC1), a fatal neurodegenerative disorder accompanied by lipid trafficking defects. In this study, we focused on the crosstalk between neurons and microglia to elucidate the mechanisms underlying extensive microgliosis in the NPC1‐affected brain. Microglia in the OB of NPC1 mice strongly expressed CX3C chemokine receptor 1 (Cx3cr1), a specific receptor for the neural chemokine C‐X3‐C motif ligand 1 (Cx3cl1). In addition, a high level of Cx3cl1 was detected in NPC1 mouse‐derived CSF due to enhanced catalytic activity of Cathepsin S (Ctss), which is responsible for Cx3cl1 secretion. Notably, nasal delivery of Cx3cl1 neutralizing antibody or Ctss inhibitor could inhibit the Cx3cl1–Cx3cr1 interaction and support neuronal survival through the suppression of microglial activation, leading to an improvement in the olfactory function in NPC1 mice. Relevant in vitro experiments revealed that intracellular cholesterol accumulation could act as a strong inducer of abnormal Ctss activation and, in turn, stimulated the Cx3cl1–Cx3cr1 axis in microglia via p38 mitogen‐activated protein kinase signaling. Our data address the significance of Cx3cl1–Cx3cr1 interaction in the development of microglial neurotoxicity and suggest that Ctss is a key upstream regulator. Therefore, this study contributes to a better understanding of the crosstalk between neurons and microglia in the development of the neurodegeneration and provides a new perspective for the management of olfactory deficits and other microglia‐dependent neuropathies. GLIA 2016;64:2291–2305

Human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis via regulation of B lymphocyte maturation

Mesenchymal stem cell (MSC) has been applied for the therapy of allergic disorders due to its beneficial immunomodulatory abilities. However, the underlying mechanisms for therapeutic efficacy are reported to be diverse according to the source of cell isolation or the route of administration. We sought to investigate the safety and the efficacy of human adipose tissue-derived MSCs (hAT-MSCs) in mouse atopic dermatitis (AD) model and to determine the distribution of cells after intravenous administration. Murine AD model was established by multiple treatment of Dermatophagoides farinae. AD mice were intravenously infused with hAT-MSCs and monitored for clinical symptoms. The administration of hAT-MSCs reduced the gross and histological signatures of AD, as well as serum IgE level. hAT-MSCs were mostly detected in lung and heart of mice within 3 days after administration and were hardly detectable at 2 weeks. All of mice administered with hAT-MSCs survived until sacrifice and did not demonstrate any adverse events. Co-culture experiments revealed that hAT-MSCs significantly inhibited the proliferation and the maturation of B lymphocytes via cyclooxygenase (COX)-2 signaling. Moreover, mast cell (MC) degranulation was suppressed by hAT-MSC. In conclusion, the intravenous infusion of hAT-MSCs can alleviate AD through the regulation of B cell function.

BMI1 inhibits senescence and enhances the immunomodulatory properties of human mesenchymal stem cells via the direct suppression of MKP-1/DUSP1

For the application of mesenchymal stem cells (MSCs) as clinical therapeutics, the regulation of cellular aging is important to protect hMSCs from an age-associated decline in their function. In this study, we evaluated the effects of hypoxia on cellular senescence and the immunomodulatory abilities of hUCB-MSCs. Hypoxic-cultured hUCB-MSCs showed enhanced proliferation and had increased immunosuppressive effects on mitogen-induced mononuclear cell proliferation. We found that BMI1, a member of the polycomb repressive complex protein group, showed increased expression in hypoxic-cultured hUCB-MSCs, and the further knock-down of BMI1 in hypoxic cells induced decreased proliferative and immunomodulatory abilities in hUCB-MSCs, along with COX-2/PGE2 down-regulation. Furthermore, the expression of phosphorylated p38 MAP kinase increased in response to the over-expression of BMI1 in normoxic conditions, suggesting that BMI1 regulates the immunomodulatory properties of hUCB-MSCs via p38 MAP kinase-mediated COX-2 expression. More importantly, we identified BMI1 as a direct repressor of MAP kinase phosphatase-1 (MKP-1)/DUSP1, which suppresses p38 MAP kinase activity. In conclusion, our results demonstrate that BMI1 plays a key role in the regulation of the immunomodulatory properties of hUCB-MSCs, and we suggest that these findings might provide a strategy to enhance the functionality of hUCB-MSCs for use in therapeutic applications.

Direct Conversion of Human Umbilical Cord Blood into Induced Neural Stem Cells with SOX2 and HMGA2

Recent advances have shown the direct reprogramming of mouse and human fibroblasts into induced neural stem cells (iNSCs) without passing through an intermediate pluripotent state. Thus, direct reprogramming strategy possibly provides a safe and homogeneous cellular platform. However, the applications of iNSCs for regenerative medicine are limited by the restricted availability of cell sources. Human umbilical cord blood (hUCB) cells hold great potential in that immunotyped hUCB units can be immediately obtained from public banks. Moreover, hUCB samples do not require invasive procedures during collection or an extensive culture period prior to reprogramming. We recently reported that somatic cells can be directly converted into iNSCs with high efficiency and a short turnaround time. Here, we describe the detailed method for the generation of iNSCs derived from hUCB (hUCB iNSCs) using the lineage-specific transcription factors SOX2 and HMGA2. The protocol for deriving iNSC-like colonies takes 1~2 weeks and establishment of homogenous hUCB iNSCs takes additional 2 weeks. Established hUCB iNSCs are clonally expandable and multipotent producing neurons and glia. Our study provides an accessible method for generating hUCB iNSCs, contributing development of in vitro neuropathological model systems.

Donor-dependent variation of human umbilical cord blood mesenchymal stem cells in response to hypoxic preconditioning and amelioration of limb ischemia

With the rapidly growing demand for mesenchymal stem cell (MSC) therapy, numerous strategies using MSCs for different diseases have been studied and reported. Because of their immunosuppressive properties, MSCs are commonly used as an allogeneic treatment. However, for the many donors who could potentially be used, it is important to understand the capacity for therapeutic usage with donor-to-donor heterogeneity. In this study, we aimed to investigate MSCs as a promising therapeutic strategy for critical limb ischemia. We evaluated MSCs from two donors (#55 and #64) and analyzed the capacity for angiogenesis through in vivo and in vitro assays to compare the therapeutic effect between different donors. We emphasized the importance of intra-population heterogeneity of MSCs on therapeutic usage by evaluating the effects of hypoxia on activating cellular angiogenesis in MSCs. The precondition of hypoxia in MSCs is known to enhance therapeutic efficacy. Our study suggests that sensitivity to hypoxic conditions is different between cells originating from different donors, and this difference affects the contribution to angiogenesis. The bioinformatics analysis of different donors under hypoxic culture conditions identified intrinsic variability in gene expression patterns and suggests alternative potential genetic factors ANGPTL4, ADM, SLC2A3, and CDON as guaranteed general indicators for further stem cell therapy.Stem cell therapy: Finding the best cellsStem cells from some donors have a stronger therapeutic effect than others, and can be identified by genetic markers. Adult or mesenchymal stem cells (MSCs), which can differentiate into many other cell types, show promise to treat many diseases. Kyung-Sun Kang at Seoul National University and co-workers investigated whether MSCs from some donors show better therapeutic efficacy than others. They tested MSCs from several donors in a mouse model simulating a stroke in a hindlimb, and measured regrowth of blood vessels. MSCs from one donor showed higher regenerative efficacy, triggering regrowth of many blood vessels and allowing the limb to be saved. Kang and co-workers used genetic analysis to identify genes that showed higher expression in these MSCs. This gene expression profile could be used to select MSCs with a higher regenerative capacity.