Osteopontin attenuates acute gastrointestinal graft-versus-host disease by preventing apoptosis of intestinal epithelial cells.

BACKGROUND AND AIMS: Acute graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation, which often targets gastrointestinal (GI) tract. Osteopontin (OPN) plays an important physiological role in the efficient development of Th1 immune responses and cell survival by inhibiting apoptosis. The role of OPN in acute GI-GVHD is poorly understood. In the present study, we investigated the role of OPN in donor T cells in the pathogenicity of acute GI-GVHD. METHODS: OPN knockout (KO) mice and C57BL/6 (B6) mice were used as donors, and (C57BL/6 × DBA/2) F1 (BDF1) mice were used as allograft recipients. Mice with acute GI-GVHD were divided into three groups: the control group (BDF1→BDF1), B6 group (B6→BDF1), and OPN-KO group (OPN-KO→BDF1). Bone marrow cells and spleen cells from donors were transplanted to lethally irradiated recipients. Clinical GVHD scores were assessed daily. Recipients were euthanized on day 7 after transplantation, and colons and small intestines were collected for various analyses. RESULTS: The clinical GVHD score in the OPN-KO group was significantly increased compared with the B6 and control groups. We observed a difference in the severity of colonic GVHD between the OPN-KO group and B6 group, but not small intestinal-GVHD between these groups. Interferon-γ, Tumor necrosis factor-α, Interleukin-17A, and Interleukin-18 gene expression in the OPN-KO group was differed between the colon and small intestine. Flow cytometric analysis revealed that the fluorescence intensity of splenic and colonic CD8 T cells expressing Fas Ligand was increased in the OPN-KO group compared with the B6 group. CONCLUSION: We demonstrated that the importance of OPN in T cells in the onset of acute GI-GVHD involves regulating apoptosis of the intestinal cell via the Fas-Fas Ligand pathway.

Establishment of a refined culture method for rat colon organoids.

BACKGROUND: Methods for the artificial three-dimensional (3D) culture of mouse and human small-intestinal and large-intestinal stem cells have been established with CD24+ or Paneth cell niches. In contrast, no studies have established stable 3D culture for rat colon stem cells. In this study, we established an advanced method for efficient rat colonic stem cell culture. METHODS: Using various tissue homogenates, we investigated the colonic organoid forming capacity under the TMDU protocol immediately adjacent to Ootani's 3D culture assembly in the same culture dish. Next, we examined whether the supernatant from the colon could be replaced by a colon homogenate. Finally, we identified the bioactive substances that were indispensable for efficient organoid culture using protein purification by three-step column chromatography and proteomic analysis with a quantitative nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By combining Ootani's method with the TMDU protocol, we established a refined culture method for Lewis rat colon organoids, which we refer to as the modified TMDU protocol. Furthermore, we confirmed that PGE2 and galection-4 promoted rat colonic organoid formation. CONCLUSIONS: We established efficient rat colonic stem cell cultures in vitro. This success will contribute to the study of rat intestinal-disease models.

Mesenchymal stem cell therapy ameliorates diabetic hepatocyte damage in mice by inhibiting infiltration of bone marrow-derived cells.

UNLABELLED: Although mesenchymal stem cells (MSCs) have been implicated in hepatic injury, the mechanism through which they contribute to diabetic liver disease has not been clarified. In this study, we investigated the effects of MSC therapy on diabetic liver damage with a focus on the role of bone-marrow-derived cells (BMDCs), which infiltrate the liver, and elucidated the mechanism mediating this process. Rat bone-marrow (BM)-derived MSCs were administered to high-fat diet (HFD)-induced type 2 diabetic mice and streptozotocin (STZ)-induced insulin-deficient diabetic mice. MSC-conditioned medium (MSC-CM) was also administered to examine the trophic effects of MSCs on liver damage. Therapeutic effects of MSCs were analyzed by assessing serum liver enzyme levels and histological findings. Kinetic and molecular profiles of BMDCs in the liver were evaluated using BM-chimeric mice. Curative effects of MSC and MSC-CM therapies were similar because both ameliorated the aggravation of aspartate aminotransferase and alanine aminotransferase at 8 weeks of treatment, despite persistent hyperlipidemia and hyperinsulinemia in HFD-diabetic mice and persistent hyperglycemia in STZ-diabetic mice. Furthermore, both therapies suppressed the abnormal infiltration of BMDCs into the liver, reversed excessive expression of proinflammatory cytokines in parenchymal cells, and regulated proliferation and survival signaling in the liver in both HFD- and STZ-diabetic mice. In addition to inducing hepatocyte regeneration in STZ-diabetic mice, both therapies also prevented excessive lipid accumulation and apoptosis of hepatocytes and reversed insulin resistance (IR) in HFD-diabetic mice. CONCLUSION: MSC therapy is a powerful tool for repairing diabetic hepatocyte damage by inhibiting inflammatory reactions induced by BMDCs and IR. These effects are likely the result of humoral factors derived from MSCs.

Mesenchymal stem cells cancel azoxymethane-induced tumor initiation.

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O(6)-methylguanine (O(6) MeG) adducts through O(6) MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-ß signaling because such properties were completely abrogated by absorption of TGF-ß under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-ß-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease.

Standardization of robot-assisted pelvic lymph node dissection-Development of a common understanding of regional anatomy and surgical technique based on cross-disciplinary discussion among colorectal surgery, urology, and gynecology.

BACKGROUND: Pelvic lymph node dissection is a procedure performed in gastroenterological surgery, urology, and gynecology. However, due to discrepancies in the understanding of pelvic anatomy among these departments, cross-disciplinary discussions have not been easy. Recently, with the rapid spread of robotic surgery, the importance of visual information in understanding pelvic anatomy has become even more significant. In this project, we attempted to clarify a shared understanding of pelvic anatomy through cross-disciplinary discussions. METHOD: From May 2020 to November 2021, a total of 11 discussions were held entirely online with 5 colorectal surgery specialists, 4 urologists, and 4 gynecologists. The discussions focused on evidence from each specialty and surgical videos, aiming to create a universally understandable pelvic anatomical illustration. RESULTS: The common area of dissection recognized across the three departments was identified as the obturator lymph nodes. A dynamic illustration of pelvic anatomy was created. In addition to a bird's-eye view of the pelvis, a pelvic half view was developed to enhance understanding of the deeper pelvic anatomy. The following insights were incorporated into the illustration: (1) the cardinal ligament in gynecology partly overlaps with the vesicohypogastric fascia in colorectal surgery; (2) the obturator lymph nodes continue cephalad into the fossa of Marcille in urology; and (3) the deep uterine vein in gynecology corresponds to the inferior vesical vein in colorectal surgery. CONCLUSION: Based on the dynamic illustration of pelvic anatomy from cross-disciplinary discussions, we anticipate advancements in pelvic lymph node dissection aiming for curative and safe outcomes.

Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow.

Hematopoietic stem cells (HSCs) are maintained, and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and nondiabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, ß-catenin, and N-cadherin on LT-HSCs and ß1-integrin, ß-catenin, angiopoietin-1, and CXCL12 on OPN cells. In an in vitro coculture system, LT-HSCs isolated from nondiabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to nondiabetic OPN cells, the decreased mRNA expressions of Tie2, ß-catenin, and N-cadherin were restored to normal levels. The effects of diabetic or nondiabetic OPN cells on LT-HSCs shown in this coculture system were confirmed by the coinjection of LT-HSCs and OPN cells into bone marrow of irradiated nondiabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities and suggest that the replacement of OPN cells may represent a novel treatment strategy.

Fusion of bone marrow-derived cells with renal tubules contributes to renal dysfunction in diabetic nephropathy.

Although diabetic nephropathy (DN) is a major cause of end-stage renal disease, the mechanism of dysfunction has not yet been clarified. We previously reported that in diabetes proinsulin-producing bone marrow-derived cells (BMDCs) fuse with hepatocytes and neurons. Fusion cells are polyploidy and produce tumor necrosis factor (TNF)-α, ultimately causing diabetic complications. In this study, we assessed whether the same mechanism is involved in DN. We performed bone marrow transplantation from male GFP-Tg mice to female C57BL/6J mice and produced diabetes by streptozotocin (STZ) or a high-fat diet. In diabetic kidneys, massive infiltration of BMDCs and tubulointerstitial injury were prominent. BMDCs and damaged tubular epithelial cells were positively stained with proinsulin and TNF-α. Cell fusion between BMDCs and renal tubules was confirmed by the presence of Y chromosome. Of tubular epithelial cells, 15.4% contain Y chromosomes in STZ-diabetic mice, 8.6% in HFD-diabetic mice, but only 1.5% in nondiabetic mice. Fusion cells primarily expressed TNF-α and caspase-3 in diabetic kidney. These in vivo findings were confirmed by in vitro coculture experiments between isolated renal tubular cells and BMDCs. It was concluded that cell fusion between BMDCs and renal tubular epithelial cells plays a crucial role in DN.

Alteration of antral and proximal colonic motility induced by chronic psychological stress involves central urocortin 3 and vasopressin in rats.

Because of the difficulties in developing suitable animal models, the pathogenesis of stress-induced functional gastrointestinal disorders is not well known. Here we applied the communication box technique to induce psychological stress in rats and then examined their gastrointestinal motility. We measured upper and lower gastrointestinal motility induced by acute and chronic psychological stress and examined the mRNA expression of various neuropeptides in the hypothalamus. Chronic psychological stress disrupted the fasted motility in the antrum and accelerated motility in the proximal colon. mRNA expression of AVP, oxytocin, and urocortin 3 was increased by chronic psychological stress. Intracerebroventricular (ICV) injection of urocortin 3 disrupted the fasted motility in the antrum, while ICV injection of Ucn3 antiserum prevented alteration in antral motility induced by chronic psychological stress. ICV injection of AVP accelerated colonic motility, while ICV injection of SSR 149415, a selective AVP V1b receptor antagonist, prevented alteration in proximal colonic motility induced by chronic psychological stress. Oxytocin and its receptor antagonist L 371257 had no effect on colonic motility in either the normal or chronic psychological stress model. These results suggest that chronic psychological stress induced by the communication box technique might disrupt fasted motility in the antrum via urocortin 3 pathways and accelerates proximal colonic motility via the AVP V1b receptor in the brain.

Dilemma of physician-mothers faced with an increased home burden and clinical duties in the hospital during the COVID-19 pandemic.

PURPOSE: Since December 2019, coronavirus disease 2019 (COVID-19) has spread rapidly across the world. During the pandemic, physicians in our hospital have had to respond both to the issue of treating the patients and the increasing domestic burden associated with social disruption. The purpose of this study was to assess how much the burden on our doctors, especially female doctors, was increasing. MATERIAL AND METHODS: The Physicians' Career Support Committee in Sapporo Medical University conducted a questionnaire survey. The questionnaire inquired about a wide range of subjects with regard to working style and family life during the first and second waves of the COVID-19 pandemic, and was sent to all medical/dental physicians working in Sapporo Medical University. RESULTS: A total of 266 (42.7%) physicians in our hospital responded to our questionnaire and the data for 264 data were analyzed. The total numbers of males, females, and others, including those who did not want to specify, were 178 (67.4%), 82 (31.0%), and 4 (1.5%), respectively. Among them, 62 (23.5%) and 23 (8.7%) answered that their domestic burden was slightly or markedly increased. The increase in the domestic burden showed a significant difference between genders (p = 0.04). Even after correction for background differences using multivariate analysis, being female (p<0.001), having child dependents (p<0.001), and treating COVID-19 patients (p = 0.03) were significantly related to an increased domestic burden. Regarding family style, 58.1% of the physician-fathers were from two-income families (i.e., families with both parents in employment), and they answered that their partner mainly cared for the children. In contrast, 97.3% of physician-mothers were from two-income families, and 94.6% of the physician-mothers had to take care of children by themselves. CONCLUSION: Physician-mothers are caught in a dilemma between an increased home burden and clinical duties in the hospital, with a significantly higher ratio than physician-fathers during the pandemic. As we showed, female doctors could have not continued their careers and take responsible positions in the same way as male doctors. This is a social risk in the timing of a crisis, such as a pandemic.

Enhancing epithelial engraftment of rat mesenchymal stem cells restores epithelial barrier integrity.

The cellular origin, in vivo function and fate of donor bone marrow-derived cells residing in the recipient intestinal epithelial cells, pericryptal myofibroblasts or endothelial cells remain obscure. Although 'immunoprivileged' mesenchymal stem cells (MSCs) are prime candidates for cell- and gene-based therapy, their precise role in colitis remains largely undetermined. Using a dextran sulphate sodium (DSS) colitis with busulphan (BU)-induced hypoplastic marrow model, we examined the therapeutic effects of MSC transplantation, focusing on the role of MSCs as both cell providers and immunomodulators. Donor-derived MSCs were detected by eGFP immunofluorescence and fluorescence in situ hybridization for Y-chromosome (Y-FISH) analysis. Western blot analysis of apical-most tight junction proteins was performed with antibodies against claudin-2, -7, -8, -12, -13, -15 and ZO-1. Cytokine and cell cycle profiles were analysed by semi-quantitative RT-PCR and flow cytometry. Susceptibility to DSS colitis was significantly increased by co-existing BU-induced bone marrow hypoplasia and this increase was significantly reduced by enhancing epithelial engraftment of MSCs, an effect depending on restoring epithelial barrier integrity rather than inhibiting host immune responses. We provide evidence that implicates MSCs in maintaining epithelial barrier function by reassembling apical-most tight junction proteins, claudins. The therapeutic efficacy of extrinsic MSCs depends on enhancing epithelial engraftment in damaged crypts by busulphan conditioning. Such a role for the MSC-derived intestinal cells in colitis therapy merits further examination and may offer a promising new treatment for inflammatory bowel disease (IBD).

Down-regulation of RalGTPase-Activating Protein Promotes Colitis-Associated Cancer via NLRP3 Inflammasome Activation.

BACKGROUND & AIMS: Ral guanosine triphosphatase-activating protein α2 (RalGAPα2) is the major catalytic subunit of the negative regulators of the small guanosine triphosphatase Ral, a member of the Ras subfamily. Ral regulates tumorigenesis and invasion/metastasis of some cancers; however, the role of Ral in colitis-associated cancer (CAC) has not been investigated. We aimed to elucidate the role of Ral in the mechanism of CAC. METHODS: We used wild-type (WT) mice and RalGAPα2 knockout (KO) mice that showed Ral activation, and bone marrow chimeric mice were generated as follows: WT to WT, WT to RalGAPα2 KO, RalGAPα2 KO to WT, and RalGAPα2 KO to RalGAPα2 KO mice. CAC was induced in these mice by intraperitoneal injection of azoxymethane followed by dextran sulfate sodium intake. Intestinal epithelial cells were isolated from colon tissues, and we performed complementary DNA microarray analysis. Cytokine expression in normal colon tissues and CAC was analyzed by quantitative polymerase chain reaction. RESULTS: Bone marrow chimeric mice showed that immune cell function between WT mice and RalGAPα2 KO mice was not significantly different in the CAC mechanism. RalGAPα2 KO mice had a significantly larger tumor number and size and a significantly higher proportion of tumors invading the submucosa than WT mice. Higher expression levels of matrix metalloproteinase-9 and matrix metalloproteinase-13 were observed in RalGAPα2 KO mice than in WT mice. The expression levels of interleukin 1ß, NLRP3, apoptosis associated speck-like protein containing a CARD, and caspase-1 were apparently increased in the tumors of RalGAPα2 KO mice compared with WT mice. NLRP3 inhibitor reduced the number of invasive tumors. CONCLUSIONS: Ral activation participates in the mechanism of CAC development via NLRP3 inflammasome activation.

Mitochondria transfer from mesenchymal stem cells structurally and functionally repairs renal proximal tubular epithelial cells in diabetic nephropathy in vivo.

The underlying therapeutic mechanism of renal tubular epithelium repair of diabetic nephropathy (DN) by bone marrow-derived mesenchymal stem cells (BM-MSCs) has not been fully elucidated. Recently, mitochondria (Mt) transfer was reported as a novel action of BM-MSCs to rescue injured cells. We investigated Mt transfer from systemically administered BM-MSCs to renal proximal tubular epithelial cells (PTECs) in streptozotocin (STZ)-induced diabetic animals. BM-MSCs also transferred their Mt to impaired PTECs when co-cultured in vitro, which suppressed apoptosis of impaired PTECs. Additionally, BM-MSC-derived isolated Mt enhanced the expression of mitochondrial superoxide dismutase 2 and Bcl-2 expression and inhibited reactive oxygen species (ROS) production in vitro. Isolated Mt also inhibited nuclear translocation of PGC-1α and restored the expression of megalin and SGLT2 under high glucose condition (HG) in PTECs. Moreover, isolated Mt directly injected under the renal capsule of STZ rats improved the cellular morphology of STZ-PTECs, and the structure of the tubular basement membrane and brush border in vivo. This study is the first to show Mt transfer from systemically administered BM-MSCs to damaged PTECs in vivo, and the first to investigate mechanisms underlying the potential therapeutic effects of Mt transfer from BM-MSCs in DN.

An enriched environment prevents diabetes-induced cognitive impairment in rats by enhancing exosomal miR-146a secretion from endogenous bone marrow-derived mesenchymal stem cells.

Increasing evidence suggests that an enriched environment (EE) ameliorates cognitive impairment by promoting repair of brain damage. However, the mechanisms by which this occurs have not been determined. To address this issue, we investigated whether an EE enhanced the capability of endogenous bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to prevent hippocampal damage due to diabetes by focusing on miRNA carried in BM-MSC-derived exosomes. In diabetic streptozotocin (STZ) rats housed in an EE (STZ/EE), cognitive impairment was significantly reduced, and both neuronal and astroglial damage in the hippocampus was alleviated compared with STZ rats housed in conventional cages (STZ/CC). BM-MSCs isolated from STZ/CC rats had functional and morphological abnormalities that were not detected in STZ/EE BM-MSCs. The miR-146a levels in exosomes in conditioned medium of cultured BM-MSCs and serum from STZ/CC rats were decreased compared with non-diabetic rats, and the level was restored in STZ/EE rats. Thus, the data suggest that increased levels of miR-146a in sera were derived from endogenous BM-MSCs in STZ/EE rats. To examine the possibility that increased miR-146a in serum may exert anti-inflammatory effects on astrocytes in diabetic rats, astrocytes transfected with miR-146a were stimulated with advanced glycation end products (AGEs) to mimic diabetic conditions. The expression of IRAK1, NF-κB, and tumor necrosis factor-α was significantly higher in AGE-stimulated astrocytes, and these factors were decreased in miR-146a-transfected astrocytes. These results suggested that EEs stimulate up-regulation of exosomal miR-146a secretion by endogenous BM-MSCs, which exerts anti-inflammatory effects on damaged astrocytes and prevents diabetes-induced cognitive impairment.

Umbilical cord extracts improve osteoporotic abnormalities of bone marrow-derived mesenchymal stem cells and promote their therapeutic effects on ovariectomised rats.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the most valuable source of autologous cells for transplantation and tissue regeneration to treat osteoporosis. Although BM-MSCs are the primary cells responsible for maintaining bone metabolism and homeostasis, their regenerative ability may be attenuated in postmenopausal osteoporosis patients. Therefore, we first examined potential abnormalities of BM-MSCs in an oestrogen-deficient rat model constructed by ovariectomy (OVX-MSCs). Cell proliferation, mobilisation, and regulation of osteoclasts were downregulated in OVX-MSCs. Moreover, therapeutic effects of OVX-MSCs were decreased in OVX rats. Accordingly, we developed a new activator for BM-MSCs using human umbilical cord extracts, Wharton's jelly extract supernatant (WJS), which improved cell proliferation, mobilisation and suppressive effects on activated osteoclasts in OVX-MSCs. Bone volume, RANK and TRACP expression of osteoclasts, as well as proinflammatory cytokine expression in bone tissues, were ameliorated by OVX-MSCs activated with WJS (OVX-MSCs-WJ) in OVX rats. Fusion and bone resorption activity of osteoclasts were suppressed in macrophage-induced and primary mouse bone marrow cell-induced osteoclasts via suppression of osteoclast-specific genes, such as Nfatc1, Clcn7, Atp6i and Dc-stamp, by co-culture with OVX-MSCs-WJ in vitro. In this study, we developed a new activator, WJS, which improved the functional abnormalities and therapeutic effects of BM-MSCs on postmenopausal osteoporosis.

Activated forms of astrocytes with higher GLT-1 expression are associated with cognitive normal subjects with Alzheimer pathology in human brain.

Although the cognitive impairment in Alzheimer's disease (AD) is believed to be caused by amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs), several postmortem studies have reported cognitive normal subjects with AD brain pathology. As the mechanism underlying these discrepancies has not been clarified, we focused the neuroprotective role of astrocytes. After examining 47 donated brains, we classified brains into 3 groups, no AD pathology with no dementia (N-N), AD pathology with no dementia (AD-N), and AD pathology with dementia (AD-D), which represented 41%, 21%, and 38% of brains, respectively. No differences were found in the accumulation of Aß plaques or NFTs in the entorhinal cortex (EC) between AD-N and AD-D. Number of neurons and synaptic density were increased in AD-N compared to those in AD-D. The astrocytes in AD-N possessed longer or thicker processes, while those in AD-D possessed shorter or thinner processes in layer I/II of the EC. Astrocytes in all layers of the EC in AD-N showed enhanced GLT-1 expression in comparison to those in AD-D. Therefore these activated forms of astrocytes with increased GLT-1 expression may exert beneficial roles in preserving cognitive function, even in the presence of Aß and NFTs.

Umbilical cord extracts improve diabetic abnormalities in bone marrow-derived mesenchymal stem cells and increase their therapeutic effects on diabetic nephropathy.

Bone marrow-derived mesenchymal stem cells (BM-MSC) has been applied as the most valuable source of autologous cell transplantation for various diseases including diabetic complications. However, hyperglycemia may cause abnormalities in intrinsic BM-MSC which might lose sufficient therapeutic effects in diabetic patients. We demonstrated the functional abnormalities in BM-MSC derived from both type 1 and type 2 diabetes models in vitro, which resulted in loss of therapeutic effects in vivo in diabetic nephropathy (DN). Then, we developed a novel method to improve abnormalities in BM-MSC using human umbilical cord extracts, namely Wharton's jelly extract supernatant (WJs). WJs is a cocktail of growth factors, extracellular matrixes and exosomes, which ameliorates proliferative capacity, motility, mitochondrial degeneration, endoplasmic reticular functions and exosome secretions in both type 1 and type 2 diabetes-derived BM-MSC (DM-MSC). Exosomes contained in WJs were a key factor for this activation, which exerted similar effects to complete WJs. DM-MSC activated by WJs ameliorated renal injury in both type 1 and type 2 DN. In this study, we developed a novel activating method using WJs to significantly increase the therapeutic effect of BM-MSC, which may allow effective autologous cell transplantation.

Bone marrow-derived mesenchymal stem cells improve diabetes-induced cognitive impairment by exosome transfer into damaged neurons and astrocytes.

The incidence of dementia is higher in diabetic patients, but no effective treatment has been developed. This study showed that rat bone marrow mesenchymal stem cells (BM-MSCs) can improve the cognitive impairments of STZ-diabetic mice by repairing damaged neurons and astrocytes. The Morris water maze test demonstrated that cognitive impairments induced by diabetes were significantly improved by intravenous injection of BM-MSCs. In the CA1 region of the hippocampus, degeneration of neurons and astrocytes, as well as synaptic loss, were prominent in diabetes, and BM-MSC treatment successfully normalized them. Since a limited number of donor BM-MSCs was observed in the brain parenchyma, we hypothesized that humoral factors, especially exosomes released from BM-MSCs, act on damaged neurons and astrocytes. To investigate the effectiveness of exosomes for treatment of diabetes-induced cognitive impairment, exosomes were purified from the culture media and injected intracerebroventricularly into diabetic mice. Recovery of cognitive impairment and histological abnormalities similar to that seen with BM-MSC injection was found following exosome treatment. Use of fluorescence-labeled exosomes demonstrated that injected exosomes were internalized into astrocytes and neurons; these subsequently reversed the dysfunction. The present results indicate that exosomes derived from BM-MSCs might be a promising therapeutic tool for diabetes-induced cognitive impairment.

Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes.

Bone marrow-derived mesenchymal stem cells (MSCs) have contributed to the improvement of diabetic nephropathy (DN); however, the actual mediator of this effect and its role has not been characterized thoroughly. We investigated the effects of MSC therapy on DN, focusing on the paracrine effect of renal trophic factors, including exosomes secreted by MSCs. MSCs and MSC-conditioned medium (MSC-CM) as renal trophic factors were administered in parallel to high-fat diet (HFD)-induced type 2 diabetic mice and streptozotocin (STZ)-induced insulin-deficient diabetic mice. Both therapies showed approximately equivalent curative effects, as each inhibited the exacerbation of albuminuria. They also suppressed the excessive infiltration of BMDCs into the kidney by regulating the expression of the adhesion molecule ICAM-1. Proinflammatory cytokine expression (e.g., TNF-α) and fibrosis in tubular interstitium were inhibited. TGF-ß1 expression was down-regulated and tight junction protein expression (e.g., ZO-1) was maintained, which sequentially suppressed the epithelial-to-mesenchymal transition of tubular epithelial cells (TECs). Exosomes purified from MSC-CM exerted an anti-apoptotic effect and protected tight junction structure in TECs. The increase of glomerular mesangium substrate was inhibited in HFD-diabetic mice. MSC therapy is a promising tool to prevent DN via the paracrine effect of renal trophic factors including exosomes due to its multifactorial action.

Stem cell therapy for inflammatory bowel disease.

Inflammatory bowel disease (IBD) could be curable by "immune rest" and correction of the genetic predisposition inherent in allogeneic hematopoietic stem cell transplantation. However, balancing risks against benefits remains challenging. The application of mesenchymal stem cells (MSCs) serving as a site-regulated "drugstore" is a recent concept, which suggests the possibility of an alternative treatment for many intractable diseases such as IBD. Depending on the required function of MSC, such as a cell provider, immune moderator, and/or trophic resource, MSC therapy should be optimized to maximize its therapeutic benefit. Therapeutic effects do not always require full engraftment of MSCs. Therefore, optimization of pleiotropic gut trophic factors produced by MSCs, which favoring not only regulating immune responses but also promoting tissue repair, must directly enhance new drug discoveries for treatment of IBD. Stem cell biology holds great promise for a new era of cell-based therapy, sparking considerable interest among scientists, clinicians, and patients. However, the translational arm of stem cell science remains in a relatively primitive state. Although several clinical studies using MSCs have been initiated, early results suggest several inherent problems. In each study, optimization of MSC therapy appears to be the most urgent problem, and can be resolved only by scientifically unveiling the mechanisms of therapeutic action. In the present review, the authors outline how such information would facilitate the critical steps in the paradigm shift from basic research on stem cell biology to clinical practice of regenerative medicine for conquering IBD in the near future.