Human Adipose-Derived Mesenchymal Stromal Cells Exhibit High HLA-DR Levels and Altered Cellular Characteristics under a Xeno-free and Serum-free Condition.

BACKGROUND: We have observed an increased expression of negative markers in some clinical-grade, xeno- and serum-free cultured adipose-derived mesenchymal stem/stromal cell (ADMSC) samples. It gave rise to concern that xeno- and serum-free conditions might have unexpected effects on human ADMSCs. This study aims to test this hypothesis for two xeno- and serum-free media, PowerStem MSC1 media (PS) and StemMACS MSC Expansion Media (SM), that support the in vitro expansion of ADMSCs. METHODS: We investigated the expression of negative markers in 42 clinical-grade ADMSC samples expanded in PS. Next, we cultured ADMSCs from seven donors in PS and SM and examined their growth and colony-forming ability, surface marker expression, differentiation, cell cycle and senescence, as well as genetic stability of two passages representing an early and late passage for therapeutic MSCs. RESULTS: 15 of 42 clinical-grade PS-expanded ADMSC samples showed an increased expression of negative markers ranging from 2.73% to 34.24%, which positively correlated with the age of donors. This rise of negative markers was related to an upregulation of Human Leukocyte Antigen - DR (HLA-DR). In addition, the PS-cultured cells presented decreased growth ability, lower frequencies of cells in S/G2/M phases, and increased ß-galactosidase activity in passage 7 suggesting their senescent feature compared to those grown in SM. Although MSCs of both PS and SM cultures were capable of multilineage differentiation, the PS-cultured cells demonstrated chromosomal abnormalities in passage 7 compared to the normal karyotype of their SM counterparts. CONCLUSIONS: These findings suggest that the SM media is more suitable for the expansion of therapeutic ADMSCs than PS. The study also hints a change of ADMSC features at more advanced passages and with increased donor's age. Thus, it emphasizes the necessity to cover these aspects in the quality control of therapeutic MSC products.

Type 2 diabetes mellitus duration and obesity alter the efficacy of autologously transplanted bone marrow-derived mesenchymal stem/stromal cells.

Human bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) represent promising stem cell therapy for the treatment of type 2 diabetes mellitus (T2DM), but the results of autologous BM-MSC administration in T2DM patients are contradictory. The purpose of this study was to test the hypothesis that autologous BM-MSC administration in T2DM patient is safe and that the efficacy of the treatment is dependant on the quality of the autologous BM-MSC population and administration routes. T2DM patients were enrolled, randomly assigned (1:1) by a computer-based system into the intravenous and dorsal pancreatic arterial groups. The safety was assessed in all the treated patients, and the efficacy was evaluated based on the absolute changes in the hemoglobin A1c, fasting blood glucose, and C-peptide levels throughout the 12-month follow-up. Our data indicated that autologous BM-MSC administration was well tolerated in 30 T2DM patients. Short-term therapeutic effects were observed in patients with T2DM duration of <10 years and a body mass index <23, which is in line with the phenotypic analysis of the autologous BM-MSC population. T2DM duration directly altered the proliferation rate of BM-MSCs, abrogated the glycolysis and mitochondria respiration of BM-MSCs, and induced the accumulation of mitochondria DNA mutation. Our data suggest that autologous administration of BM-MSCs in the treatment of T2DM should be performed in patients with T2DM duration <10 years and no obesity. Prior to further confirming the effects of T2DM on BM-MSC biology, future work with a larger cohort focusing on patients with different T2DM history is needed to understand the mechanism underlying our observation.

Can Autologous Adipose-Derived Mesenchymal Stem Cell Transplantation Improve Sexual Function in People with Sexual Functional Deficiency?

BACKGROUND: Sexual functional deficiency occurs at some point in life and becomes a problematic issue in middle-aged adulthood. Regenerative medicine, especially mesenchymal stem cell (MSC) transplantation, has developed extensively, with preclinical and clinical trials emphasizing the benefits of stem cell therapy for restoration of sexual deficiency. This study was designed to develop a new therapeutic stem cell treatment for people with sexual functional deficiency. METHODS: Thirty-one patients, including 15 males and 16 females with a medical history of reduced sexual activity, met the inclusion criteria and were enrolled in the study, phase I/IIa clinical trial with a 12-month follow-up. Adipose tissue-derived mesenchymal stem/stromal cells (ADSC) were isolated by type I collagenase digestion and cultured at the Stem Cell Core Facility under ISO 14644-1. Each participant received 1 million cells/kg of body weight via the intravenous route. Safety was evaluated by assessing the occurrence of adverse events or severe adverse events. Efficacy was assessed in males by monitoring testosterone levels and administering the International Index of Erectile Function (IIEF) questionnaire and in females by monitoring anti-Mullerian hormone (AMH), estradiol (E2), and follicle-stimulating hormone (FSH) levels and administering the Female Sexual Functioning Index (FSFI) questionnaire at baseline and 3-, 6-, and 12-months post-transplantation. RESULTS: There was no occurrence of severe adverse events after ADSC administration in our study. Post-transplantation sexual satisfaction was observed in all patients enrolled in this study. Testosterone levels in males increased soon after transplantation and were maintained at high levels for up to 6 months before decreasing again at the 12-month follow-up. No significant changes in AMH, FSH or E2 levels were recorded in female patients. CONCLUSIONS: Autologous ADSC infusion is a potential therapeutic option for patients with reduced sexual activity, especially for male patients. TRIAL REGISTRATION: ClinicalTrials.gov. NCT03346967, Registered November 20, 2017.

Outcomes of bone marrow mononuclear cell transplantation combined with interventional education for autism spectrum disorder.

The aim of this study was to evaluate the safety and efficacy of autologous bone marrow mononuclear cell transplantation combined with educational intervention for children with autism spectrum disorder. An open-label clinical trial was performed from July 2017 to August 2019 at Vinmec International Hospital, Hanoi, Vietnam. Thirty children who fulfilled the autism criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, and had Childhood Autism Rating Scale (CARS) scores >37 were selected. Bone marrow was harvested by anterior iliac crest puncture under general anesthesia. The volume collected was as follows: 8 mL/kg for patients under 10 kg (80 mL + [body weight in kg - 10] × 7 mL) for patients above 10 kg. Mononuclear cells were isolated with a Ficoll gradient and then infused intrathecally. The same procedure was repeated 6 months later. After the first transplantation, all patients underwent 8 weeks of educational intervention based on the Early Start Denver Model. There were no severe adverse events associated with transplantation. The severity of autism spectrum disorder (ASD) was significantly reduced, with the median CARS score decreasing from 50 (range 40-55.5) to 46.5 (range 33.5-53.5) (P < .05). Adaptive capacity increased, with the median Vineland Adaptive Behavior Scales score rising from 53.5 to 60.5. Social communication, language, and daily skills improved markedly within 18 months after transplantation. Conversely, repetitive behaviors and hyperactivity decreased remarkably. Autologous bone marrow mononuclear cell transplantation in combination with behavioral intervention was safe and well tolerated in children with ASD (Trial registration: ClinicalTrials.gov identifier: NCT03225651).

Expression of aldo-keto reductase family 1, member C14 during ovulation in the rat.

The potent androgen 5α-dihydrotestosterone is metabolized to the weak androgen 5α-androstane-3α, 17ß-diol (3α-diol) by the enzyme aldo-keto reductase family 1, member C14 (Akr1c14) in rodents. The purpose of the present study was to investigate the regulation of Akr1c14 expression during the ovulatory process in rat ovaries. Northern blot analysis revealed that treatment of immature rats with equine chorionic gonadotropin resulted in lowered Akr1c14 expression, whereas subsequent treatment with human chorionic gonadotropin (hCG) increased ovarian Akr1c14 expression within 3 h. In situ hybridization analysis showed that Akr1c14 mRNA was localized in granulosa cells of growing follicles before hCG treatment, but it was also expressed in granulosa cells of preovulatory follicles after hCG treatment. Akr1c14 protein expression increased after 6 h of hCG treatment and was sustained at high levels until 12 h. The levels of 3α-diol in preovulatory follicles isolated from ovaries in vivo were fluctuated by hCG treatment; decreased at 6 h and increased at 9 h. Human CG-induced Akr1c14 expression was suppressed by treatment with the progesterone receptor antagonist RU486, but not with the cyclooxygenase inhibitor indomethacin. Taken together, these findings demonstrate the induction of Akr1c14 by hCG in granulosa cells of rat preovulatory follicles that was regulated by progesterone receptor antagonist.

Regulation of interleukin-11 expression in ovulatory follicles of the rat ovary.

The aim of the present study was to examine the regulation of interleukin (IL)-11 expression, as well as the role of IL-11, during ovulation in gonadotropin-primed immature rats. Injection of equine chorionic gonadotropin (eCG), followed by human CG (hCG) to induce superovulation stimulated expression of the Il11 gene in theca cells within 6h, as revealed by northern blot and in situ hybridisation analyses. Real-time reverse transcription-polymerase chain reaction analysis showed that the IL-11 receptor, α subunit gene was expressed in granulosa and theca cells and that injection of hCG had no effect on its expression. IL-11 protein expression was stimulated in theca cells by hCG. LH-stimulated increases in Il11 mRNA levels in cultured preovulatory follicles were inhibited by protein kinase A and mitogen-activated protein kinase kinase inhibitors. Toll-like receptor (TLR) 2 and TLR4 were detected in preovulatory follicles, and the TLR4 ligand lipopolysaccharide, but not the TLR2 ligand Pam3Cys, increased Il11 mRNA levels in theca cells, but not in granulosa cells. Treatment of preovulatory follicles with IL-11 stimulated progesterone production and steroidogenic acute regulatory protein (Star) gene expression. Together, these results indicate that IL-11 in theca cells is stimulated by mitogen-activated protein kinase signalling and TLR4 activation, and increases progesterone production during ovulation.

Regulation of uridine diphosphate-glucuronosyltransferase 2B15 expression during ovulation in the rat.

Uridine diphosphate-glucuronosyltransferase 2B15 (UGT2B15) conjugates 5α-androstane-3α, 17ß-diol (3α-diol) to 3α-diol glucuronide (3α-diol G) in steroid target tissues. The present study investigated the regulation of UGT2B15 expression during the ovulatory process in the rat. Real-time PCR analysis revealed that treatment of immature rats with equine chorionic gonadotropin followed by human chorionic gonadotropin transiently stimulated UGT2B15 gene expression in granulosa cells of preovulatory follicles within 6 h. The progesterone receptor antagonist RU486 suppressed the gonadotropin-induced UGT2B15 expression. The expression of UGT2B15 and the levels of 3α-diol G were transiently increased by luteinizing hormone (LH) treatment in cultured preovulatory follicles. The LH-stimulated UGT2B15 mRNA level in cultured preovulatory follicles was inhibited by inhibitors of adenylyl cyclase, phosphoinositide 3-kinase and mitogen-activated protein kinase. Furthermore, a vitamin D receptor agonist (calcitriol) suppressed the LH-stimulated UGT2B15 expression in a dose-dependent manner. Taken together, these results indicate that gonadotropins transiently stimulate UGT2B15 expression and activity in preovulatory follicles, and UGT2B15 mRNA levels are regulated by the progesterone receptor and vitamin D receptor.

Cumulus cell-expressed type I interferons induce cumulus expansion in mice.

Ovulation resembles the inflammatory response. The purpose of the present study was to examine the expression and role of type I interferons (IFNs) Ifnalpha and Ifnbeta in mouse ovaries during the process of ovulation. An in vivo injection of equine chorionic gonadotropin (CG)-human CG (hCG) stimulated Ifnalpha and Ifnbeta mRNA in cumulus-oocyte complexes (COCs) within 6 h. Type I IFN receptor (Ifnar1 and Ifnar2) genes were also expressed in preovulatory follicles without a change by hCG. Immunofluorescent study revealed the expression of protein signals of Ifnalpha, Ifnbeta, and Ifnar1 in cumulus cells. Treatment of COCs with Ifnalpha or Ifnbeta in vitro induced cumulus expansion that was comparable to that mediated by epiregulin. In cultured COCs, the levels of Ifnalpha and Ifnbeta mRNA increased by epiregulin and follicle-stimulating hormone, but not by prostaglandin E2. Ifnalpha and Ifnbeta activated multiple signaling events (signal transducer and activator of transcription-1/3, Akt, and mitogen-activated protein kinase 1/2) and stimulated the expression of genes known to impact COC expansion (Has2, Ptx3, Tnfaip6, and Ptgs2). Interestingly, treatment of COCs with Toll-like receptor (TLR) 2 and TLR4 ligands (lipopolysaccharides, Pam3Cys, and hyaluronan fragments) increased Ifnalpha and Ifnbeta mRNA, while coculture with anti-TLR2/4 neutralizing antibody abolished these effects. Taken together, these results demonstrate that the type I IFN system is operating in mouse cumulus cells and plays a role in the induction of cumulus expansion during the ovulatory process in mice.