Injection Of Adipose-derived Regenerative Cells Research Articles

RTA-dh404 decreased oxidative stress in mice ischemic limbs and augmented efficacy of therapeutic angiogenesis by intramuscular injection of adipose-derived regenerative cells in the limbs

Although an intramuscular injection of angiogenic cells to ischemic limbs with peripheral artery disease is a therapeutic option to rescue patients by augmenting neovascularization in the limbs, oxidative stress in the limbs may accelerate apoptosis of the injected cells and thereby reduce the therapeutic effect. In this study involving mice with ischemic lower limbs, whether daily oral administration of RTA-dh404, which is an activator of nuclear factor erythroid 2-related factor 2 (Nrf2) with antioxidant activity, could reduce oxidative stress in the limbs and suppress apoptosis of adipose-derived regenerative cells (ADRCs) injected in the limbs, eventually augmenting neovascularization in the limbs, was evaluated. The tissue expression of Nrf2 and concentrations of total antioxidant capacity and superoxide dismutase in the mice ischemic limbs were higher in the RTA-dh404-treated mice than in the control treated mice, and oxidative stress in the limbs of the RTA-dh404 treated mice was decreased. The day after an intramuscular injection of human ADRCs into ischemic lower limbs of immunodeficient mice, the number of apoptotic ADRCs in the ischemic limbs was decreased by approximately 25% in the RTA-dh404-treated mice compared to the control mice. Fourteen days after cell injection, neovascularization and the salvage ratio were increased by approximately 10% and 63%, respectively, in the ischemic limbs in the RTA-dh404-treated mice compared to the control mice. Pretreatment of ischemic limbs by daily oral administration of RTA-dh404 may augment the effect of therapeutic angiogenesis using an intramuscular injection of ADRCs into the ischemic limbs.

Influence of background characteristics in responders of regenerative therapy by periurethral injection of adipose-derived regenerative cells for male stress urinary incontinence.

To determine if the male responders with post-prostatectomy incontinence in the ADRESU study, which is a clinical trial of regenerative therapy by periurethral injection of adipose-derived regenerative cells, are influenced by any background characteristics. Briefly, autologous adipose-derived regenerative cells isolated from abdominal adipose tissue and a mixture of adipose-derived regenerative cells with fat tissue were transurethrally injected into the rhabdosphincter and submucosal space of the urethra, respectively. Sixteen out of 43 patients (37.2%) responded to treatment (responders) and exhibited improvement in the urine leakage volume, defined as >50% reduction from baseline determined by the 24-hour pad test at 52 weeks of treatment (or last visit within 52 weeks). Background data such as age, body weight, method of prostatectomy, baseline frequency of leaks, number of leaks, number of pad changes, International Consultation on Incontinence Questionnaire-Short Form, King's Health Questionnaire, urodynamic urethral function including functional profile length and maximum urethral closure pressure, and abdominal leak point pressure were collected and compared between responders and nonresponders. None of the background factors influenced improvement in the responders as compared with the nonresponders. However, a significant between-group difference in the rates of decrease in urine leakage volume was noted in patients of younger age (<70 years), compared with those of older age (≥70 years) from 2 to 26 weeks of treatment. A greater decrease in urine leakage volume was noted in the younger patient group than in the older patient group.

Abstract 13303: Identification of Critical Six Transcriptional Factors for Reprogramming of Mouse Adipose-Derived Regenerative Cells Toward Cardiomyocytes and Its Therapeutic Effect

Background: Adipose-derived regenerative cells (ADRCs) are promising stem cells for regenerative medicine that are easy to harvest from adipose tissues and can expand in vitro . While ADRCs can differentiate into mesodermal lineage cells including adipocytes, osteocytes, and chondrocytes, little is known about differentiating adult ADRCs into mature cardiomyocytes. Objective: The aim of this study was to explore how to differentiate adult mouse ADRCs into cardiomyocytes efficiently and test their effect on the myocardial infarction mouse model in vivo . Method and Results: We performed differential expression analysis to identify transcriptional factors which were upregulated in developing mice heart (E11.5) compared with ADRCs. We systemically screened the combinations of the required transcription factors in ADRCs derived from αMHC-EGFP reporter mice. Here, we identified the following six genes; Baf60c , Gata4 , Gata6 , Klf15 , Mef2a , Myocd , not more or less, as the best combination to efficiently induce αMHC in ADRCs. Fluorescence-activated cell sorting analysis showed that 3.0±0.61% (n=3) ADRCs were positive for αMHC after introducing these six factors. In addition, quantitative PCR demonstrated that several other marker genes of mature cardiomyocytes, including Actc1 , Tnnt2 , Ttn , Fhod3 , were confirmed to be upregulated by six factors. Immunocytochemical staining showed that most of the induced ADRCs had fibrous conformation of sarcomeric α-actin. Moreover, the gene expression pattern of the cells showed upregulation of genes associated with cardiac muscle cell differentiation. Finally, injection of the induced ADRCs into acute myocardial infarction lesions in vivo resulted in the improvement of survival rate (p=0.028, n=32), fractional shortening (p=0.012, n=7, 15), and the reduction of infarction scar area compared to the injection of ADRCs without induction (p=0.029, n=7). The induced ADRCs could survive in situ and continued to express the cardiac troponin T protein for four weeks after injection. Conclusions: The combination of six transcriptional factors could induce cardiomyocyte-like gene expression patterns on adult ADRCs. The induced ADRCs could have a therapeutic effect on an experimental ischemic heart model in vivo .

Attenuation of Knee Osteoarthritis Progression in Mice through Polarization of M2 Macrophages by Intra-Articular Transplantation of Non-Cultured Human Adipose-Derived Regenerative Cells.

Adipose-derived regenerative cells (ADRCs) are non-cultured heterogeneous or mixed populations of cells obtained from adipose tissue by collagenase digestion. The injection of ADRCs have been tried clinically for the treatment of osteoarthritis (OA). The purpose of this study was to evaluate the effect of intra-articular transplantation of human ADRCs on OA progression in mice and the effect of ADRCs on macrophage polarization. In in vivo experiments, BALB/c-nu mice with knee OA received intra-articular transplantation of either phosphate buffered-saline or human ADRCs. OA progression was evaluated histologically and significantly attenuated in the ADRC group at both four and eight weeks postoperatively. The expression of OA-related proteins in the cartilage and macrophage-associated markers in the synovium were examined by immunohistochemistry. The numbers of MMP-13-, ADAMTS-5-, IL-1β-, IL-6- and iNOS-positive cells significantly decreased, and type II collagen- and CD206-positive cells were more frequently detected in the ADRC group compared with that in the control group. In vitro co-culture experiments showed that ADRCs induced macrophage polarization toward M2. The results of this study suggest that the intra-articular transplantation of human ADRCs could attenuate OA progression possibly by reducing catabolic factors in chondrocytes and modulating macrophage polarization.

Abstract 12889: Clathrin-mediated Endocytosis Plays a Key Role in Stem Cell Therapy for Ischemic Heart Disease

Background and Aim: Cell therapy represents a potential frontier to transform the treatment and prognosis of heart failure and acute myocardial infarction (AMI). Extracellular vesicles (EVs) have come out as key mediators of intercellular communication to improve cardiac function in cell therapy. However, the mechanisms by which cardiomyocytes respond to EVs remain unclear. Here, we sought to clarify the effect of cardiomyocyte endocytosis on AMI. Methods and Results: Treatment with the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under conditions of hypoxia in vitro. In vivo, the injection of ADRCs in the heart simultaneous with coronary artery ligation decreased infarct area in the heart in a dose-dependent manner. In addition, the injection of ADRCs prevented cardiac rupture after AMI and significantly reduced apoptosis in cardiomyocytes at 1 and 2 days. To examine the efficacy of cardiomyocyte endocytosis, cardiomyocytes were cultured with EVs previously isolated from ADRC culture supernatant and labeled with the fluorescent dye, PKH67. Notably, hypoxic culture conditions increased the intensity of the labeled EVs in cardiomyocytes. An inhibitor of clathrin-mediated endocytosis (chlorpromazine), but not inhibitors of caveolae-mediated endocytosis, micropinocytosis, and phagocytosis (genistein, cytochalasin D, or amiloride), significantly suppressed the ADRC culture supernatant-induced decreases of hypoxia-damaged cardiomyocytes. Chlorpromazine treatment also decreased hypoxia-induced cardiomyocyte capture of labeled EVs secreted from ADRCs. To investigate whether communication between cardiomyocytes and ADRCs affords cardiac protection through clathrin-mediated endocytosis in vivo, mice were treated with ADRCs alone or in combination with chlorpromazine following coronary artery ligation. Chlorpromazine treatment failed to show ADRC-induced cardiac protection such as prevention of cardiac rupture and anti-apoptotic effect. Conclusions: Our results indicate that EVs secreted from ADRCs prevent cardiac rupture after AMI and that clathrin-mediated endocytosis plays a critical role in transporting the EVs into cardiomyocytes.

No influence on tumor growth by intramuscular injection of adipose-derived regenerative cells: safety evaluation of therapeutic angiogenesis with cell therapy.

Therapeutic angiogenesis with autologous stem/progenitor cells is a promising novel strategy for treatment of severe ischemic diseases. Human clinical trials utilizing autologous adipose-derived regenerative cells (ADRCs) have not reported treatment-related critical adverse effects thus far. However, there is still a large knowledge gap regarding whether treatment of ischemic diseases with angiogenic therapy using ADRCs would promote unfavorable angiogenesis associated with tumors in vivo. Herein, we addressed this clinical question using a mouse hindlimb ischemia (HLI) and simultaneous remote tumor implantation model. C57BL/6J background wild-type mice were injected with murine B16F10 melanoma cells on their back, 1 day before ischemic surgery. These mice were subjected to surgical unilateral hindlimb ischemia, followed by ADRC implantation or PBS injection into the hindlimb ischemic muscles on the next day. Intramuscular implantation of ADRCs enhanced tissue capillary density and blood flow examined by a laser Doppler blood perfusion analysis in hind limb. However, this therapeutic regimen for ischemic limb using ADRCs did not affect remote melanoma growth nor the density of its feeder artery, angiogenesis, and lymphatic vessels compared with the PBS group. In addition, no distant metastases were detected in any of the mice regardless of the group. In conclusion, local implantation of ADRCs promotes angiogenesis in response to tissue ischemia in the hindlimb without promoting remote tumor growth and related angio/lymphangiogenesis. Therapeutic angiogenesis to the ischemic hindlimb using ADRCs seems to be safe regarding remote tumor growth.NEW & NOTEWORTHY In this study, we demonstrated that local injection of ADRCs can promote angiogenesis in response to tissue ischemia without promoting remote tumor growth in a mouse model. Our findings indicate that therapeutic angiogenesis to the ischemic hindlimb using ADRCs seems to be safe regarding remote tumor growth.

Therapeutic effect of adipose-derived regenerative cells on bladder function in rats with underactive bladder.

ABSTRACTWe examined the effect of adipose-derived regenerative cells (ADRC) on bladder functions in a rat model of detrusor underactivity (DU) induced by bladder over-distention.Adult female Sprague Dawley rats were divided into 3 groups: sham group (control); over-distention group; and over-distention with ADRC treatment group. Bladder was over-distended with saline (2.7mL) on day 1, 8, 15 and 22 of the study. ADRCs, which were harvested from male F344 rats, expanded via culture, were injected into the bladder wall at day 15. Cystometry and in vitro organ bath functional studies were performed on day 28. Moreover, histological assessment of the bladder was performed.In cystometry, significant prolongation of the inter-contraction interval (ICI) and decrease of voiding efficiency (VE) were observed in the over-distention group, compared to that in the control group. Significant improvement in ICI and VE was seen in the ADRC treatment group in comparison with the over-distention group. The over-distention group showed significantly weaker bladder contractile responses to carbachol and electrical field stimulation than the control group, while bladder contractile responses were significantly stronger in the ADRC treatment group than that in the over-distention group. The over-distention group showed substantial fibrosis of the bladder compared to the control group, whereas bladder fibrosis was alleviated in the ADRC treatment group.In conclusion, the injection of ADRC into bladder wall improved bladder dysfunction and histological changes induced by bladder over-distention. ADRCs-based regenerative therapy could be novel treatment for DU.

Second-look arthroscopic findings of cartilage and meniscus repair after injection of adipose-derived regenerative cells in knee osteoarthrits: Report of two cases.

BackgroundThe purpose of this study was to use second-look arthroscopic findings and clinical assessment to determine outcome in two cases of knee osteoarthritis treated by intra-articular knee injection of adipose-derived regenerative cells (ADRCs).Case presentationThis study involved two patients who received ADRC therapy for knee osteoarthritis and completed the six-month post-treatment follow-up period. For each treatment, 130 mL of subcutaneous adipose tissue was harvested using tumescent liposuction technique and manual aspiration of tissue from the thigh using a suction cannula under local anesthesia in the operating room. The adipose tissue harvested was processed using the Celution® Centrifuge in a dedicated cell processing room. The ADRCs were injected into the articular cavity of both knees for one patient and into a single affected knee in the second patient (three joints). Pain and knee function were assessed using a Visual Analogue Scale (VAS) and the Knee Outcome in Osteoarthritis Score (KOOS) respectively. The cartilage defect was assessed by direct visualization (arthroscopy). No serious adverse events were reported throughout follow-up. Pain and knee function were significantly improved from baseline in all treated knees at one, three and six months after ADRCs. At six-months after ADRCs treatment, the second-look arthroscopy showed that almost all the cartilage defect areas were covered by regenerated cartilage, some cartilage fibrillation area was reduced, and meniscus tear areas were repaired.ConclusionsCartilage and meniscus repair were observed six-months after ADRCs therapy under second-look arthroscopy. It was shown that a single administration of ADRCs might be effective as a treatment for knee osteoarthritis.

Cardiomyocytes capture stem cell-derived, anti-apoptotic microRNA-214 via clathrin-mediated endocytosis in acute myocardial infarction

Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication that have the potential to improve cardiac function when used in cell-based therapy. However, the means by which cardiomyocytes respond to EVs remains unclear. Here, we sought to clarify the role of exosomes in improving cardiac function by investigating the effect of cardiomyocyte endocytosis of exosomes from mesenchymal stem cells on acute myocardial infarction (MI). Exposing cardiomyocytes to the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under hypoxia in vitro. In vivo, the injection of ADRCs into the heart simultaneous with coronary artery ligation decreased overall cardiac infarct area and prevented cardiac rupture after acute MI. Quantitative RT-PCR-based analysis of the expression of 35 known anti-apoptotic and secreted microRNAs (miRNAs) in ADRCs revealed that ADRCs express several of these miRNAs, among which miR-214 was the most abundant. Of note, miR-214 silencing in ADRCs significantly impaired the anti-apoptotic effects of the ADRC treatment on cardiomyocytes in vitro and in vivo To examine cardiomyocyte endocytosis of exosomes, we cultured the cardiomyocytes with ADRC-derived exosomes labeled with the fluorescent dye PKH67 and found that hypoxic culture conditions increased the levels of the labeled exosomes in cardiomyocytes. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, significantly suppressed the ADRC-induced decrease of hypoxia-damaged cardiomyocytes and also decreased hypoxia-induced cardiomyocyte capture of both labeled EVs and extracellular miR-214 secreted from ADRCs. Our results indicate that clathrin-mediated endocytosis in cardiomyocytes plays a critical role in their uptake of circulating, exosome-associated miRNAs that inhibit apoptosis.

Lipografting and stromal vascular fractions of adipose tissue in the treatment of patients with radiation-induced rectovaginal fistulas

Objective : to analyze the efficacy of a newly developed method for the treatment of radiation-induced rectovaginal fistulas using autologous adipose tissue obtained by aspiration liposuction. Materials and methods . This retrospective study included 24 patients with radiation-induced rectovaginal fistulas that arose after chemoradiotherapy for malignant tumors of the female reproductive system. All patients underwent colostomy before treatment initiation. Study participants received 1 to 5 injections (mean number 3) of autologous adipose-derived regenerative cells. The main indication for the procedure was the presence of a fistula, diagnosed at least 6 months after the last course of radiation therapy. We evaluated time to fistula healing, results of elastometry, and presence of complications. Results. The mean total dose received by patients during their radiotherapy was 60 Gy; median time to initiation of fistula treatment was 15 months. Before the injection of adipose-derived regenerative cells, the mean fistula diameter was 2.50 + 0.46 cm, whereas after 3 months, it reduced to 1.35 + 0.47 cm and after 6 months, it was 1.12 + 0.47 cm. All patients had complete epithelialization of the defect. No cases of fistula recurrence were registered. Conclusion. We demonstrated high efficacy of a new reconstructive method for radiation-induced rectovaginal fistulas with autologous adipose tissue obtained by aspiration liposuction. Its effectiveness is confirmed by complete fistula closure and restoration of intestinal continuity in all study participants.

Treatment of male stress urinary incontinence using autologous adipose-derived regenerative cells: Long-term efficacy and safety.

To investigate the long-term efficacy and safety of periurethral injection of autologous adipose-derived regenerative cells for the treatment of post-prostatectomy stress urinary incontinence. A total of 13 patients with persistent stress urinary incontinence after prostate surgery (radical prostatectomy, 10 patients; holmium laser enucleation of the prostate, three patients) underwent periurethral injection of adipose-derived regenerative cells and were followed up for >4years. A 24-h pad test was carried out for four consecutive days in each evaluation period, and changes in the mean daily leakage volume during the 4days from baseline to 60months after treatment were evaluated. The mean follow-up period was 69months (range 55-72months). The mean leakage volume/24h in all patients changed from 260.7g to 152.7g. Urinary incontinence progressively improved up to 12months after treatment in 10 patients, who maintained improvement up to the final assessment, with the mean daily leakage volume decreasing from 281.5g to 119.0g (reduction rate 57.7%). The other three patients showed no improvement at 1year and at the final assessment. After the perioperative period, significant adverse events or prostate-specific antigen increase were not observed during long-term follow up. The present findings suggest that periurethral injection of autologous adipose-derived regenerative cells is a safe and feasible treatment modality with long-term efficacy for patients with male stress urinary incontinence caused by urethral sphincter deficiency.

Adipose-Derived Regenerative Cells

IntroductionStudies in our hybrid rat model of transplantation/ischemic reperfusion (IRI) have demonstrated improvement in kidney function post injection of adipose-derived regenerative cells (ADRCs) into the renal artery. The mechanism on how these cells establish regenerative conditions during IRI

Design of a single-arm clinical trial of regenerative therapy by periurethral injection of adipose-derived regenerative cells for male stress urinary incontinence in Japan: the ADRESU study protocol

BackgroundMale stress urinary incontinence is a prevalent condition after radical prostatectomy. While the standard recommendation for the management of urine leakage is pelvic floor muscle training, its efficacy is still unsatisfactory. Therefore, we have focused on regenerative therapy, which consists of administering a periurethral injection of autologous regenerative cells from adipose tissue, separated using the Celution® system. Based on an interim data analysis of our exploratory study, we confirmed the efficacy and acceptable safety profile of this treatment. Accordingly, we began discussions with Japanese regulatory authorities regarding the development of this therapy in Japan. The Ministry of Health, Labour and Welfare suggested that we implement a clinical trial of a new medical device based on the Pharmaceutical Affaires Act in Japan. Next, we discussed the design of this investigator-initiated clinical trial (the ADRESU study) aimed at evaluating the efficacy and safety of this therapy, in a consultation meeting with the Pharmaceuticals and Medical Device Agency.MethodsThe ADRESU study is an open-label, multi-center, single-arm study involving a total of 45 male stress urinary incontinence patients with mild-to-moderate urine leakage persisting more than 1 year after prostatectomy, in spite of behavioral and pharmacological therapies. The primary endpoint is the rate of patients at 52 weeks with improvement of urine leakage volume defined as a reduction from baseline greater than 50% by 24-h pad test. Our specific hypothesis is that the primary endpoint result will be higher than a pre-specified threshold of 10%.DiscussionThe ADRESU study is the first clinical trial of regenerative treatment for stress urinary incontinence by adipose-derived regenerative cells using the Celution® system based on the Japanese Pharmaceutical Affaires Act. We will evaluate the efficacy and safety in this trial to provide an adequate basis for marketing approval with the final objective of making this novel therapy widely available for Japanese patients.Trial registrationThis trial was registered at the University Hospital Medical information Network Clinical Trial Registry (UMIN-CTR Unique ID: UMIN000017901; Registered July 1, 2015) and at ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT02529865; Registered August 18, 2015).