Mesangial cell regeneration from exogenous stromal progenitor by utilizing embryonic kidney.

Kidney regenerative medicine is expected to be the solution to the shortage of organs for transplantation. In a previous report, we transplanted exogenous renal progenitor cells (RPCs) including nephron progenitor cells (NPCs), stromal progenitor cells (SPCs), and the ureteric bud (UB) into the nephrogenic zone of animal embryos and succeeded in regenerating new nephrons from exogenous NPCs through a fetal developmental program. However, it was unknown whether the renal stromal lineage cells were regenerated from SPCs. The present study aimed to verify the differentiation of SPCs into mesangial cells and renal stromal lineage cells. Here, we found that simply transplanting RPCs, including SPCs, into the nephrogenic zone of wild-type fetal mice was insufficient for differentiation of SPCs. Therefore, to enrich the purity of SPCs, we sorted cells from RPCs by targeting platelet-derived growth factor receptor alpha (PDGFRa) which is a cell surface marker for immature stromal cells and transplanted the PDGFRa-positive sorted cells. As a result, we succeeded in regenerating a large number of mesangial cells and other renal stromal lineage cells including interstitial fibroblasts, vascular pericytes, and juxtaglomerular cells. We have established the method for regeneration of stromal cells from exogenous SPCs that may contribute to various fields, such as regenerative medicine and kidney embryology, and the creation of disease models for renal stromal disorders.

Mesenchymal-Epithelial Transition in Culture of Stromal Progenitor Cells Isolated from the Liver of a Patient with Alcoholic Cirrhosis.

The cells isolated from biopsy specimen of a patient with alcoholic liver cirrhosis and cultured under standard conditions for obtaining stromal cell culture clearly diverged during early passages into two morphologically and phenotypically different subtypes: epithelial and mesenchymal. Mesenchymal cells expressed CD90 and CD44 and epithelial cells expressed CD166, CD227, and hepatocyte growth factor receptor Met. Starting from passage 6, the culture underwent spontaneous morphological changes and by passages 8-10 contained only epithelium-like cells. CD90 and CD44 expression disappeared, CD166 and CD227 expression remained unchanged, and Met expression increased. A small fraction of cells expressed GATA-4, HNF3ß, HNF1α, and HNF4α. After addition of inducers of hepatogeneic differentiation, the cells started producing albumin.

Natural compounds and mesenchymal stem cells: implications for inflammatory-impaired tissue regeneration.

Inflammation is a common and important pathological process occurring in any part of the body and relating to a variety of diseases. Effective tissue repair is critical for the survival of impaired organisms. Considering the side effects of the currently used anti-inflammatory medications, new therapeutic agents are urgently needed for the improvement of regenerative capacities of inflammatory-impaired tissues. Mesenchymal stromal stem/progenitor cells (MSCs) are characterized by the capabilities of self-renewal and multipotent differentiation and exhibit immunomodulatory capacity. Due to the ability to modulate inflammatory phenotypes and immune responses, MSCs have been considered as a potential alternative therapy for autoimmune and inflammatory diseases. Natural compounds (NCs) are complex small multiple-target molecules mostly derived from plants and microorganisms, exhibiting therapeutic effects in many disorders, such as osteoporosis, diabetes, cancer, and inflammatory/autoimmune diseases. Recently, increasing studies focused on the prominent effects of NCs on MSCs, including the regulation of cell survival and inflammatory response, as well as osteogenic/adipogenic differentiation capacities, which indicate the roles of NCs on MSC-based cytotherapy in several inflammatory diseases. Their therapeutic effects and fewer side effects in numerous physiological processes, compared to chemosynthetic drugs, made them to be a new therapeutic avenue combined with MSCs for impaired tissue regeneration. Here we summarize the current understanding of the influence of NCs on MSCs and related downstream signaling pathways, specifically in pathological inflammatory conditions. In addition, the emerging concepts through the combination of NCs and MSCs to expand the therapeutic perspectives are highlighted. A promising MSC source from oral/dental tissues is also discussed, with a remarkable potential for MSC-based therapy in future clinical applications.

Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication.

Aberrant CpG-island methylation affects ovarian cancer progression. The promotor methylation changes at tumor suppressive genes in ovarian cancer stromal progenitor cells (OCSPCs) and epithelial ovarian cancer (EOC) tissues and their clinical implication remains unexplored. We systemically analyzed the promoter methylation status of 40 tumor suppressor genes (TSGs) associated with cancer in paired epithelial-like and mesenchymal-like OCSPCs and ovarian cancer cells by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The effect of DNA methylation on gene expression was confirmed using qRT-PCR. The differential frequencies of TSGs' promoter methylation among matched epithelial-like or mesenchymal-like OCSPCs from tissues and ascites and ovarian cancer tissues were further validated in cancer tissues and correlated with clinicopathological features and survival outcomes of patients. According to the promoter methylation frequencies of the 40 TSGs, promoters of RASSF1A were the only significantly hypomethylated in epithelial-like OCSPCs from tissues than those from ascites and bulk tumor cells (0% vs 38% vs 45%, P=0.039 by Fisher's exact test). The most frequencies at promotor hypermethylation of TSGs in mesenchymal-like OCSPCs from ascites which processed aggressiveness were CDKN2B (73%) followed by CCND2 (45%) and RASSF1A (45%). Forty-three percent (47/110) of RASSF1A and 45% of CCND2 were validated as a frequently hypermethylated gene in an independent set of 110 EOC tissues in contrast to none (0/60) and 12% (10/60) of benign ovarian cysts (both P<0.001). Functional experiments revealed overexpression of CCND2 or CDKN2B in MSc-OCSPCs decreases EMT, invasion, and spheroid formation in EOC, and abolishes DNMT1 and COL6A3 expression. However, for the expected 5-year overall survival (OS) for patients with methylated RASSF1A, CCND2, and CDKN2B, only RASSF1A was significantly worse than those without methylated RASSF1A (56% vs 80%, p=0.022). Taken together, overexpression of CCND2 and CDKN2B decreased the aggressiveness of mesenchymal-like OCSPCs from ascites which may represent a potential therapeutic target for EOC. Promotor hypomethylation at RASSF1A in OCSPCs from EOC tissues and changes to hypermethylation of EOC and OCSPCs from ascites could predict poor survival outcomes for EOC patients compared to without those changes of CCND2 and CDKN2B.

Osteogenic potential of gingival stromal progenitor cells cultured in platelet rich fibrin is predicted by core-binding factor subunit-α1/Sox9 expression ratio ( in vitro).

Background: Alveolar bone defect regeneration has long been problematic in the field of dentistry. Gingival stromal progenitor cells (GSPCs) offer a promising solution for alveolar bone regeneration. In order to optimally differentiate and proliferate progenitor cells, growth factors (GFs) are required. Platelet rich fibrin (PRF) has many GFs and can be easily manufactured. Core-binding factor subunit-α1 (CBF-α1) constitutes a well-known osteogenic differentiation transcription factor in SPCs. Sox9, as a chondrogenic transcription factor, interacts and inhibits CBF-α1, but its precise role in direct in vitro osteogenesis remains unknown. GSPCs cultured in vitro in PRF to optimally stimulate osteogenic differentiation has been largely overlooked. The aim of this study was to analyze GSPCs cultured in PRF osteogenic differentiation predicted by CBF-α1/Sox9. Methods: This study used a true experimental with post-test only control group design and random sampling. GPSCs isolated from the lower gingiva of four healthy, 250-gram, 1-month old, male Wistar rats ( Rattus Novergicus) were cultured for two weeks, passaged every 4-5 days. GSPCs in passage 3-5 were cultured in five M24 plates (N=108; n=6/group) for Day 7, Day 14, and Day 21 in three different mediums (control negative group: αModified Eagle Medium; control positive group: High Glucose-Dulbecco's Modified Eagle Medium (DMEM-HG) + osteogenic medium; Treatment group: DMEM-HG + osteogenic medium + PRF). CBF-α1 and Sox9 were examined with ICC monoclonal antibody. A one-way ANOVA continued with Tukey HSD test (p<0.05) based on Kolmogorov-Smirnov and Levene's tests (p>0.05) was performed. Results: The treatment group showed the highest CBF-α1/Sox9 ratio (16.00±3.000/14.33±2.517) on Day 7, while the lowest CBF-α1/Sox9 ratio (3.33±1.528/3.67±1.155) occurred in the control negative group on Day 21, with significant difference between the groups (p<0.05). Conclusion: GSPCs cultured in PRF had potential osteogenic differentiation ability predicted by the CBF-α1/sox9 ratio.

CD146 (MCAM) in human cs-DLK1-/cs-CD34+ adipose stromal/progenitor cells.

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1-/cs-CD34+/cs-CD146- ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146- cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31-/CD34-/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31-/CD34+/CD146+). Additionally, we detected wavy CD34-/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34-/CD146+ cells. Both CD34-/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34-/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1-/cs-CD34+/cs-CD146- ASCs.

Stromal Progenitor Cells in Mitigation of Non-Hematopoietic Radiation Injuries.

PURPOSE OF REVIEW: Therapeutic exposure to high doses of radiation can severely impair organ function due to ablation of stem cells. Normal tissue injury is a dose-limiting toxicity for radiation therapy (RT). Although advances in the delivery of high precision conformal RT has increased normal tissue sparing, mitigating and therapeutic strategies that could alleviate early and chronic radiation effects are urgently needed in order to deliver curative doses of RT, especially in abdominal, pelvic and thoracic malignancies. Radiation-induced gastrointestinal injury is also a major cause of lethality from accidental or intentional exposure to whole body irradiation in the case of nuclear accidents or terrorism. This review examines the therapeutic options for mitigation of non-hematopoietic radiation injuries. RECENT FINDINGS: We have developed stem cell based therapies for the mitigation of acute radiation syndrome (ARS) and radiation-induced gastrointestinal syndrome (RIGS). This is a promising option because of the robustness of standardized isolation and transplantation of stromal cells protocols, and their ability to support and replace radiation-damaged stem cells and stem cell niche. Stromal progenitor cells (SPC) represent a unique multipotent and heterogeneous cell population with regenerative, immunosuppressive, anti-inflammatory, and wound healing properties. SPC are also known to secrete various key cytokines and growth factors such as platelet derived growth factors (PDGF), keratinocyte growth factor (KGF), R-spondins (Rspo), and may consequently exert their regenerative effects via paracrine function. Additionally, secretory vesicles such as exosomes or microparticles can potentially be a cell-free alternative replacing the cell transplant in some cases. SUMMARY: This review highlights the beneficial effects of SPC on tissue regeneration with their ability to (a) target the irradiated tissues, (b) recruit host stromal cells, (c) regenerate endothelium and epithelium, (d) and secrete regenerative and immunomodulatory paracrine signals to control inflammation, ulceration, wound healing and fibrosis.

Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells.

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue.

Sorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34-, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1-/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34- cells. Permeabilized cs-DLK1-/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31-). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34- and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34- perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31- cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity.

Immunolocalization of CD34 Positive Progenitor Cells in Diabetic and Non Diabetic Periodontitis Patients - A Comparative Study.

BACKGROUND: Little research has been documented to determine the CD34 positive cells in healthy periodontium, chronic periodontitis and in chronic periodontitis with diabetes mellitus individuals. AIM: The aim of the present study was to evaluate and compare the CD34 positive progenitor cells of the gingiva in patients with healthy periodontium, chronic periodontitis and chronic periodontitis with Diabetes Mellitus. MATERIALS AND METHODS: A total number of 75 patients were divided into 3 groups which included Group I (healthy periodontium), Group II (chronic periodontitis) and Group III (chronic periodontitis with diabetes mellitus). Periodontal examination included Plaque index, Gingival index, Gingival bleeding index, Probing pocket depth and Clinical attachment levels. Gingival biopsies were collected from each participant and they were fixed in formalin embedded in paraffin which was later subjected to immunohistochemical procedure with anti-CD34 (a stemness marker). T-Test and Regression analysis (R-square) was used to analyse the data. RESULTS: The mean number of CD34 positive cells were higher in group III (5.71±1.97) compared to Group chronic periodontitis group I (4.98± 2.08) and II (4.48± 1.24). CONCLUSION: Although CD34 is a non specific stemness marker, results suggest that there is a significant difference in the number of CD34 positive progenitor cells between Group II and Group III but no significant difference was observed between Group I, II and Group I, III.