Jinbeom Heo

The Paracrine Effects of Mesenchymal Stem Cells Stimulate the Regeneration Capacity of Endogenous Stem Cells in the Repair of a Bladder-Outlet-Obstruction-Induced Overactive Bladder

Overactive bladder (OAB), which is characterized by the sudden and uncomfortable need to urinate with or without urinary leakage, is a challenging urological condition. The insufficient efficacy of current pharmacotherapies that uses antimuscarinic agents has increased the demand for novel long-term/stable therapeutic strategies. Here, we report the superior therapeutic efficacy of using mesenchymal stem cells (MSCs) for the treatment of OAB and a novel therapeutic mechanism that activates endogenous Oct4(+) primitive stem cells. We induced OAB using bladder-outlet-obstruction (BOO) in a rat model and either administered a single transplantation of human adipose-derived MSCs or daily intravenous injections of solifenacin, an antimuscarinic agent, for 2 weeks. Within 2 weeks, both the MSC- and solifenacin-treated groups similarly demonstrated relief from BOO-induced detrusor overactivity, hypertrophic smooth muscle, and neurological injuries. In contrast with the solifenacin-treated groups, a single transplantation of MSCs improved most OAB parameters to normal levels within 4 weeks. Although the transplanted human MSCs were hardly engrafted into the damaged bladders, the bladder tissues transplanted with MSCs increased rat sequence-specific transcription of Oct4, Sox2, and Stella, which are surrogate markers for primitive pluripotent stem cells. In addition, MSCs enhanced the expression of several genes, responsible for stem cell trafficking, including SDF-1/CXCR4, HGF/cMet, PDGF/PDGFR, and VEGF/VEGFR signaling axis. These changes in gene expression were not observed in the solifenacin-treated group. Therefore, we suggest the novel mechanisms for the paracrine effect of MSCs as unleashing/mobilizing primitive endogenous stem cells, which could not only explain the long-term/stable therapeutic efficacy of MSCs, but also provide promising new therapies for the treatment of OAB.

Mesenchymal Stem Cell Therapy Alleviates Interstitial Cystitis by Activating Wnt Signaling Pathway

Interstitial cystitis (IC) is a syndrome characterized by urinary urgency, frequency, pelvic pain, and nocturia in the absence of bacterial infection or identifiable pathology. IC is a devastating disease that certainly decreases quality of life. However, the causes of IC remain unknown and no effective treatments or cures have been developed. This study evaluated the therapeutic potency of using human umbilical cord-blood-derived mesenchymal stem cells (UCB-MSCs) to treat IC in a rat model and to investigate its responsible molecular mechanism. IC was induced in 10-week-old female Sprague-Dawley rats via the instillation of 0.1 M HCl or phosphate-buffered saline (PBS; sham). After 1 week, human UCB-MSC (IC+MSC) or PBS (IC) was directly injected into the submucosal layer of the bladder. A single injection of human UCB-MSCs significantly attenuated the irregular and decreased voiding interval in the IC group. Accordingly, denudation of the epithelium and increased inflammatory responses, mast cell infiltration, neurofilament production, and angiogenesis observed in the IC bladders were prevented in the IC+MSC group. The injected UCB-MSCs successfully engrafted to the stromal and epithelial tissues and activated Wnt signaling cascade. Interference with Wnt and epidermal growth factor receptor activity by small molecules abrogated the benefits of MSC therapy. This is the first report that provides an experimental evidence of the therapeutic effects and molecular mechanisms of MSC therapy to IC using an orthodox rat animal model. Our findings not only provide the basis for clinical trials of MSC therapy to IC but also advance our understanding of IC pathophysiology.

Senescence-Associated MCP-1 Secretion Is Dependent on a Decline in BMI1 in Human Mesenchymal Stromal Cells

AIMS Cellular senescence and its secretory phenotype (senescence-associated secretory phenotype [SASP]) develop after long-term expansion of mesenchymal stromal cells (MSCs). Further investigation of this phenotype is required to improve the therapeutic efficacy of MSC-based cell therapies. In this study, we show that positive feedback between SASP and inherent senescence processes plays a crucial role in the senescence of umbilical cord blood-derived MSCs (UCB-MSCs). RESULTS We found that monocyte chemoattractant protein-1 (MCP-1) was secreted as a dominant component of the SASP during expansion of UCB-MSCs and reinforced senescence via its cognate receptor chemokine (c-c motif) receptor 2 (CCR2) by activating the ROS-p38-MAPK-p53/p21 signaling cascade in both an autocrine and paracrine manner. The activated p53 in turn increased MCP-1 secretion, completing a feed-forward loop that triggered the senescence program in UCB-MSCs. Accordingly, knockdown of CCR2 in UCB-MSCs significantly improved their therapeutic ability to alleviate airway inflammation in an experimental allergic asthma model. Moreover, BMI1, a polycomb protein, repressed the expression of MCP-1 by binding to its regulatory elements. The reduction in BMI1 levels during UCB-MSC senescence altered the epigenetic status of MCP-1, including the loss of H2AK119Ub, and resulted in derepression of MCP-1. INNOVATION Our results provide the first evidence supporting the existence of the SASP as a causative contributor to UCB-MSC senescence and reveal a so far unappreciated link between epigenetic regulation and SASP for maintaining a stable senescent phenotype. CONCLUSION Senescence of UCB-MSCs is orchestrated by MCP-1, which is secreted as a major component of the SASP and is epigenetically regulated by BMI1.

Genome-wide analysis of murine bone marrow‑derived very small embryonic-like stem cells reveals that mitogenic growth factor signaling pathways play a crucial role in the quiescence and ageing of these cells

It has been postulated that the most primitive population of stem cells, Oct4+Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs), differentiate into tissue-committed stem cells in adult mice. However, Oct4+ VSELs remain quiescent in adult tissues and do not form teratomas. In thi study, we report the characteristics of the VSEL transcriptome by gene set enrichment analysis employing a microarray database established from 20 murine bone marrow-derived, FACS-sorted VSELs in comparison with hematopoietic stem cells and embryonic stem cells. In the Oct4+ VSELs, we observed the upregulation of tissue-specific gene sets and a gene set encoding the complement-coagulation cascade. By contrast, in the VSELs, we observed the downregulation of genes involved in the UV radiation response, mRNA processing and mitogenic growth factor signaling [e.g., insulin-like growth factor-1 (IGF-1) and neurotrophic tyrosine kinase receptor A (TRKA), as well as the ERK and PI3K pathways]. Employing leading-edge subset analysis and real-time PCR assays, we observed that several genes, such as growth factor receptor-bound protein 2 (GRB2), son of sevenless homolog 1 (SOS1), SHC (Src homology 2 domain containing) transforming protein 1 (SHC1), mitogen-activated protein kinase kinase 1 (MAP2K1), v-akt murine thymoma viral oncogene homolog 3 (AKT3), ELK1, ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3), glycogen synthase kinase 3β (GSK3β) and casein kinase 2, alpha 1 polypeptide (CSNK2A1), which are involved in mitogenic growth factor signaling pathways, were commonly downregulated in the VSELs. Notably, this repression was reversed in the VSELs co-cultured over a C2C12 supportive cell-line, whereby they are induced to form VSEL-derived spheres (VSEL-DSs); thus, they are enriched, forming more differentiated stem cells. Therefore, we suggest that the repression of mitogenic growth factor signaling (e.g., through the IGF-1 receptor) may prevent uncontrolled Oct4+ VSEL proliferation and teratoma formation. Thus, restoring the responsiveness to mitogenic growth factors may be a crucial step in employing these cells in regenerative medicine.

The Therapeutic Effects of Human Mesenchymal Stem Cells Primed with Sphingosine-1 Phosphate on Pulmonary Artery Hypertension

Stem cell (SC) therapy has become a potential treatment modality for pulmonary artery hypertension (PAH), but the efficacy of human SC and priming effects have not yet been established. The mobilization and homing of hematopoietic stem cells (HSCs) are modulated by priming factors that include a bioactive lipid, sphingosine-1-phosphate (S1P), which stimulates CXCR4 receptor kinase signaling. Here, we show that priming human mesenchymal stem cells (MSCs) with S1P enhances their therapeutic efficacy in PAH. Human MSCs, similar to HSCs, showed stronger chemoattraction to S1P in transwell assays. Concomitantly, MSCs treated with 0.2 μM S1P showed increased phosphorylation of both MAPKp42/44 and AKT protein compared with nonprimed MSCs. Furthermore, S1P-primed MSCs potentiated colony forming unit-fibroblast, anti-inflammatory, and angiogenic activities of MSCs in culture. In a PAH animal model induced by subcutaneously injected monocrotaline, administration of human cord blood-derived MSCs (hCB-MSCs) or S1P-primed cells significantly attenuated the elevated right ventricular systolic pressure. Notably, S1P-primed CB-MSCs, but not unprimed hCB-MSCs, also elicited a significant reduction in the right ventricular weight ratio and pulmonary vascular wall thickness. S1P-primed MSCs enhanced the expression of several genes responsible for stem cell trafficking and angiogenesis, increasing the density of blood vessels in the damaged lungs. Thus, this study demonstrates that human MSCs have potential utility for the treatment of PAH, and that S1P priming increases the effects of SC therapy by enhancing cardiac and vascular remodeling. By optimizing this protocol in future studies, SC therapy might form a basis for clinical trials to treat human PAH.

The Molecular Nature of Very Small Embryonic-Like Stem Cells in Adult Tissues

Pluripotent stem cells (PSCs) have been considered as the most important cells in regenerative medicine as they are able to differentiate into all types of cells in the human body. PSCs have been established from several sources of embryo tissue or by reprogramming of terminally differentiated adult tissue by transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc). Interestingly, accumulating evidence has demonstrated the residence of PSCs in adult tissue and with the ability to differentiate into multiple types of tissue-committed stem cells (TCSCs). We also recently demonstrated that a population of pluripotent Oct4+ SSEA-1+Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs) resides in the adult murine bone marrow (BM) and in other murine tissue. These very small (∼3–6 μm) cells express pluripotent markers such as Oct4, Nanog, and SSEA-1. VSELs could be specified into several tissue-residing TCSCs in response to tissue/organ injury, and thus suggesting that these cells have a physiological role in the rejuvenation of a pool of TCSCs under steady-state conditions. In this review article, we discuss the molecular nature of the rare population of VSELs which have a crucial role in regulating the pluripotency, proliferation, differentiation, and aging of these cells.

Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases.

Mesenchymal stem cells protect against the tissue fibrosis of ketamine-induced cystitis in rat bladder

Abuse of the hallucinogenic drug ketamine promotes the development of lower urinary tract symptoms that resemble interstitial cystitis. The pathophysiology of ketamine-induced cystitis (KC) is largely unknown and effective therapies are lacking. Here, using a KC rat model, we show the therapeutic effects of human umbilical cord-blood (UCB)-derived mesenchymal stem cells (MSCs). Daily injection of ketamine to Sprague-Dawley rats for 2-weeks resulted in defective bladder function, indicated by irregular voiding frequency, increased maximum contraction pressure, and decreased intercontraction intervals and bladder capacity. KC bladders were characterized by severe mast-cell infiltration, tissue fibrosis, apoptosis, upregulation of transforming growth factor-β signaling related genes, and phosphorylation of Smad2 and Smad3 proteins. A single administration of MSCs (1 × 106) into bladder tissue not only significantly ameliorated the aforementioned bladder voiding parameters, but also reversed the characteristic histological and gene-expression alterations of KC bladder. Treatment with the antifibrotic compound N-acetylcysteine also alleviated the symptoms and pathological characteristics of KC bladder, indicating that the antifibrotic capacity of MSC therapy underlies its benefits. Thus, this study for the first-time shows that MSC therapy might help to cure KC by protecting against tissue fibrosis in a KC animal model and provides a foundation for clinical trials of MSC therapy.

Real-Time Monitoring of Glutathione in Living Cells Reveals that High Glutathione Levels Are Required to Maintain Stem Cell Function

Summary The core functions of stem cells (SCs) are critically regulated by their cellular redox status. Glutathione is the most abundant non-protein thiol functioning as an antioxidant and a redox regulator. However, an investigation into the relationship between glutathione-mediated redox capacity and SC activities is hindered by lack of probe. Here, we demonstrate that cyanoacrylamide-based coumarin derivatives are ratiometric probes suitable for the real-time monitoring of glutathione levels in living SCs. These probes revealed that glutathione levels are heterogeneous among subcellular organelles and among individual cells and show dynamic changes and heterogeneity in repopulating SCs depending on oxidative stress or culture conditions. Importantly, a subpopulation of SCs with high glutathione levels exhibited increased stemness and migration activities in vitro and showed improved therapeutic efficiency in treating asthma. Our results indicate that high glutathione levels are required for maintaining SC functions, and monitoring glutathione dynamics and heterogeneity can advance our understanding of the cellular responses to oxidative stress.

Improved efficacy and in vivo cellular properties of human embryonic stem cell derivative in a preclinical model of bladder pain syndrome

Interstitial cystitis/bladder pain syndrome (IC/BPS) is an intractable disease characterized by severe pelvic pain and urinary frequency. Mesenchymal stem cell (MSC) therapy is a promising approach to treat incurable IC/BPS. Here, we show greater therapeutic efficacy of human embryonic stem cell (hESC)-derived multipotent stem cells (M-MSCs) than adult bone-marrow (BM)-derived counterparts for treating IC/BPS and also monitor long-term safety and in vivo properties of transplanted M-MSCs in living animals. Controlled hESC differentiation and isolation procedures resulted in pure M-MSCs displaying typical MSC behavior. In a hydrochloric-acid instillation-induced IC/BPS animal model, a single local injection of M-MSCs ameliorated bladder symptoms of IC/BPS with superior efficacy compared to BM-derived MSCs in ameliorating bladder voiding function and histological injuries including urothelium denudation, mast-cell infiltration, tissue fibrosis, apoptosis, and visceral hypersensitivity. Little adverse outcomes such as abnormal growth, tumorigenesis, or immune-mediated transplant rejection were observed over 12-months post-injection. Intravital confocal fluorescence imaging tracked the persistence of the transplanted cells over 6-months in living animals. The infused M-MSCs differentiated into multiple cell types and gradually integrated into vascular-like structures. The present study provides the first evidence for improved therapeutic efficacy, long-term safety, and in vivo distribution and cellular properties of hESC derivatives in preclinical models of IC/BPS.