Comparison of the effect of stem cell therapy and percutaneous transluminal angioplasty on diabetic foot disease in patients with critical limb ischemia

Background aimsThe aim of our study was to compare the effect of autologous stem cell therapy (SCT) and percutaneous transluminal angioplasty (PTA) on diabetic foot disease (DFD) in patients with critical limb ischemia (CLI).MethodsThirty-one patients with DFD and CLI treated by autologous stem cells and 30 patients treated by PTA were included in the study; 23 patients with the same inclusion criteria who could not undergo PTA or SCT formed the control group. Amputation-free survival, transcutaneous oxygen pressure (TcPO₂) and wound healing were assessed over 12 months.ResultsAmputation-free survival after 6 and 12 months was significantly greater in the SCT and PTA groups compared with controls (P = 0.001 and P = 0.0029, respectively) without significant differences between the active treatment groups. Increase in TcPO₂ did not differ between SCT and PTA groups until 12 months (both Ps < 0.05 compared with baseline), whereas TcPO₂ in the control group did not change over the follow-up period. More healed ulcers were observed up to 12 months in the SCT group compared with the PTA and control groups (84 versus 57.7 versus 44.4 %; P = 0.042).ConclusionsOur study showed comparable effects of SCT and PTA on CLI, a major amputation rate that was superior to conservative therapy in patients with diabetic foot and an observable effect of SCT on wound healing. Our results support SCT as a potential promising treatment in patients with CLI and diabetic foot.     

Adipose-derived stem cells alleviate osteoporosis by enchancing osteogenesis and inhibiting adipogenesis in a rabbit model

Background aimsOsteoporosis (OP) is characterized by a reduction in bone quality, which is associated with inadequacies in bone marrow mesenchymal stromal cells (BMSCs). As an alternative cell source to BMSCs, adipose-derived stem cells (ASCs) have been investigated for bone repair because of their osteogenic potential and self-renewal capability. Nevertheless, whether autologous ASCs can be used to promote bone regeneration under osteoporotic conditions has not been elucidated.MethodsThe OP rabbit model was established by means of bilateral ovariectomy (OVX). Both BMSCs and ASCs were harvested from OVX rabbits and expanded in vitro. The effects of osteogenic-induced ASCs on the in vitro adipogenic and osteogenic capabilities of BMSCs were evaluated. Autologous ASCs were then encapsulated by calcium alginate gel and transplanted into the distal femurs of OVX rabbits (n = 12). Hydrogel without loading cells was injected into the contralateral femurs as a control. Animals were killed for investigation at 12 weeks after transplantation.ResultsOsteogenic-induced ASCs were able to promote osteogenesis and inhibit adipogenesis of osteoporotic BMSCs through activation of the bone morphogenetic protein 2/bone morphogenetic protein receptor type IB signal pathway. Local bone mineral density began to increase at 8 weeks after ASC transplantation (P < 0.05). At 12 weeks, micro–computed tomography and histological evaluation revealed more new bone formation in the cell-treated femurs than in the control group (P < 0.05).ConclusionsThis study demonstrated that ASCs could stimulate proliferation and osteogenic differentiation of BMSCs in vitro and enhance bone regeneration in vivo, which suggests that autologous osteogenic-induced ASCs might be useful to alleviate OP temporally.     

Acute hemodynamics of cardiac sympathetic denervation

IntroductionWe aimed to study the immediate hemodynamic effects of thoracoscopic bilateral cardiac sympathetic denervation (CSD) for recurrent ventricular tachycardia (VT) or VT storm.MethodWe studied a group of 18 adults who underwent bilateral thoracoscopic CSD; the blood pressure (BP) and Heart Rate (HR) were continuously monitored during the surgery and up to 6 h post-operatively.ResultsImmediately on removal of the sympathetic ganglia, the patients had a drop in both the systolic (110 mm Hg to 95.8 mm Hg, p < 0.001) and diastolic BP (69.4 mm Hg to65 mm Hg, p = 0.007) along with a drop in the HR (81.6 bpm to 61.2 bpm, p < 0.001).At 6 h after CSD, the systolic and diastolic BP did not recover significantly, while there was recovery in HR (61.2 bpm to 66 bpm, p = 0.02). There was no significant difference between those with and without left ventricular (LV) systolic dysfunction.ConclusionThe acute hemodynamic changes during the perioperative period of CSD are significant but not serious. Awareness of this is useful for peri-operative management.     

Autologous bone marrow mesenchymal stromal cell therapy for “no-option” critical limb ischemia is limited by karyotype abnormalities

BackgroundCritical limb ischemia (CLI) is the most severe manifestation of peripheral vascular disease. Revascularization is the preferred therapy, but it is not achievable in 25%–40% of patients due to diffuse anatomic distribution of the disease or medical comorbidities. No-option CLI represents an unmet medical need. Mesenchymal stromal cells (MSCs) may provide salvage therapy through their angiogenic and tissue-trophic properties. This article reports a phase 1b clinical study examining the safety and feasibility of intramuscular transplantation of autologous bone-marrow MSCs for patients with no-option CLI.MethodsTwelve patients were enrolled in the clinical trial, and nine proceeded to bone marrow aspiration and culture expansion of MSCs.ResultsA high rate of karyotype abnormality (>30%) was detected in the produced cell batches, resulting in failure of release for clinical administration. Four patients were treated with the investigational medicinal product (IMP), three with a low dose of 20 × 10⁶ MSCs and one with a mid-dose of 40 × 10⁶ MSCs. There were no serious adverse events related to trial interventions, including bone marrow aspiration, IMP injection or therapy.ConclusionsThe results of this trial conclude that an autologous cell therapy approach with MSCs for critical limb ischemia is limited by the high rate of karyotype abnormalities.     

Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation

BackgroundAdipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.MethodsNormal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated.ResultsASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia.ConclusionASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.     

Autologous bone marrow mononuclear cell therapy improves symptoms in patients with end-stage peripheral arterial disease and reduces inflammation-associated parameters

Background aimsThe purpose of this study was to evaluate the effect of autologous bone marrow mononuclear cells (BM-MNCs) on symptoms and perfusion indices in severely symptomatic patients with peripheral arterial disease (PAD) without further option for endovascular or surgical revascularization.MethodsOnly patients with severe symptomatic PAD (Fontaine class IIb-IV, Rutherford category 3–6) not amenable for revascularization were treated. Bone marrow from both cristae iliacae was harvested; MNCs were isolated by the Ficoll density-gradient method and transplanted by means of intra-arterial and intramuscular injection in the index limb. Functional (pain score, ulcer healing, maximum walking distance) and perfusion indices such as ankle-brachial-index and transcutaneous oxygen pressure were documented before and after BM-MNC therapy. Additionally, serum concentration of C-reactive protein and interleukin-6 were measured as markers of inflammation before and after BM-MNC treatment.ResultsSixteen consecutive patients (four women; mean age, 63.0 ± 13 years) were treated with a mean dose of 4.2 ± 2.2 × 10⁸ BM-MNCs. At 6 months' follow-up, ankle-brachial-index, transcutaneous oxygen pressure and maximum walking distance significantly increased, whereas C-reactive protein and interleukin-6 conversely decreased (P < 0.01 versus baseline values), resulting in 88% limb salvage, 75% pain reduction and 71% complete wound healing and/or reduction of ulcer size. One major and one minor amputation were performed, both in patients with Rutherford category 6.ConclusionsAutologous BM-MNC therapy in patients with end-stage PAD improves tissue perfusion indices and decreases markers of inflammation. If our observations could be confirmed by large-scale, randomized controlled trials, BM-MNC transplantation could become an alternative therapeutic option for patients with end-stage PAD.     

A comparative assessment of adipose‐derived stem cells from subcutaneous and visceral fat as a potential cell source for knee osteoarthritis treatment

The intra‐articular injection of adipose‐derived stem cells (ASCs) is a novel potential therapy for patients with osteoarthritis (OA). However, the efficacy of ASCs from different regions of the body remains unknown. This study investigated whether ASCs from subcutaneous or visceral adipose tissue provide the same improvement of OA. Mouse and human subcutaneous and visceral adipose tissue were excised for ASC isolation. Morphology, proliferation, surface markers and adipocyte differentiation of subcutaneous ASCs (S‐ASCs) and visceral ASCs (V‐ASCs) were analysed. A surgically induced rat model of OA was established, and 4 weeks after the operation, S‐ASCs, V‐ASCs or phosphate‐buffered saline (PBS, control) were injected into the articular cavity. Histology, immunohistochemistry and gene expression analyses were performed 6 weeks after ASC injection. The ability of ASCs to differentiate into chondrocytes was assessed by in vitro chondrogenesis, and the immunosuppressive activity of ASCs was evaluated by co‐culturing with macrophages. The proliferation of V‐ASCs was significantly greater than that of S‐ASCs, but S‐ASCs had the greater adipogenic capacity than V‐ASCs. In addition, the infracted cartilage treated with S‐ASCs showed significantly greater improvement than cartilage treated with PBS or V‐ASCs. Moreover, S‐ASCs showed better chondrogenic potential and immunosuppression in vitro. Subcutaneous adipose tissue is an effective cell source for cell therapy of OA as it promotes stem cell differentiation into chondrocytes and inhibits immunological reactions.     

Adipose-derived mesenchymal stromal cells reverse high glucose–induced reduction of angiogenesis in human retinal microvascular endothelial cells

Background aimsDiabetic retinopathy (DR) is characterized by a progressive alteration of the retinal microvasculature, arising from microaneurysms to leaky vessels and finally abnormal neovascularization. The hyperglycemia-mediated loss of pericytes is a key event in vessel degeneration causing vascular destabilization. To overcome this, mesenchymal stromal cells (MSCs) have been tested as pericyte replacement in several animal models showing repair and regeneration of DR-damaged vasculature.MethodsWe hypothesized that adipose-derived mesenchymal stromal cells (ASCs) resist high glucose–induced challenges and protect human retinal microvascular endothelial cells (HRMVECs) from glucose-mediated injury. ASCs and HRMVECs were cultured under normal-glucose (NG; 1 g/L) and high-glucose (HG; 4.5 g/L) conditions comparing their phenotype and angiogenic potential.ResultsWhereas ASCs were generally unaffected by HG, HG caused a reduction of the angiogenic potential in HRMVEC. Indeed, HG-treated HRMVECs formed fewer vascular tube structures in a basement membrane angiogenesis assay. However, this was not observed in a direct ASC and HRMVEC coculture angiogenesis assay. Increased oxidative stress levels appeared to be linked to the HG-induced reduction of angiogenesis, which could be restored by ASC-conditioned medium and antioxidant treatment.ConclusionsThese findings suggest that ASC resist HG-stress whereas endothelial cell angiogenic capacity is reduced. Thus, ASC may be potentially therapeutically active in DR by restoring angiogenic deficits in retinal endothelial cells by the secretion of proangiogenic factors. However, these data also inquire for a thorough risk assessment about the timing of the ASC-based cell therapy, which can be considered advantageous at early stage of DR, but possibly detrimental at the late neo-angiogenic stage of DR.     

Marrow changes and reduced proliferative capacity of mesenchymal stromal cells from patients with “no-option” critical limb ischemia; observations on feasibility of the autologous approach from a clinical trial

Background aimsApproximately 1 in 3 patients with critical limb ischemia (CLI) are not suitable for surgical or endovascular revascularization. Those “no-option” patients are at high risk of amputation and death. Autologous bone marrow mesenchymal stromal cells (MSCs) may provide a limb salvage option. In this study, bone marrow characteristics and expansion potentials of CLI-derived MSCs produced during a phase 1b clinical trial were compared with young healthy donor MSCs to determine the feasibility of an autologous approach. Cells were produced under Good Manufacturing Practice conditions and underwent appropriate release testing.MethodsFive bone marrow aspirates derived from patients with CLI were compared with six young healthy donor marrows in terms of number of colony-forming units–fibroblast (CFUF) and mononuclear cells. The mean population doubling times and final cell yields were used to evaluate expansion potential. The effect of increasing the volume of marrow on the CFUF count and final cell yield was evaluated by comparing 5 CLI-derived MSCs batches produced from a targeted 30 mL of marrow aspirate to five batches produced from a targeted 100 mL of marrow.ResultsCLI-derived marrow aspirate showed significantly lower numbers of mononuclear cells with no difference in the number of CFUFs when compared with healthy donors’ marrow aspirate. CLI-derived MSCs showed a significantly longer population doubling time and reduced final cell yield compared with young healthy donors' MSCs. The poor growth kinetics of CLI MSCs were not mitigated by increasing the bone marrow aspirate from 30 to 100 mL.ConclusionsIn addition to the previously reported karyotype abnormalities in MSCs isolated from patients with CLI, but not in cells from healthy donors, the feasibility of autologous transplantation of bone marrow MSCs for patients with no-option CLI is further limited by the increased expansion time and the reduced cell yield.     

Rat adipose tissue-derived stem cells attenuate peritoneal injuries in rat zymosan-induced peritonitis accompanied by complement activation

Background aimsIn patients receiving peritoneal dialysis, fungal or yeast peritonitis has a poor prognosis. In rat peritoneum with mechanical scraping, severe peritonitis can be induced by zymosan, a component of yeast (Zy/scraping peritonitis). Administration of rat adipose tissue-derived stromal cells (ASCs) potentially can improve several tissue injuries. The present study investigated whether rat ASCs could improve peritoneal inflammation in Zy/scraping peritonitis.MethodsRat ASCs were injected intraperitoneally on a daily basis in rats with Zy/scraping peritonitis.ResultsPeritoneal inflammation accompanied by accumulation of inflammatory cells and complement deposition was suppressed by day 5 after injection of rat ASCs. The peritoneal mesothelial layer in Zy/scraping peritonitis with rat ASC treatment was restored compared with the peritoneal mesothelial layer without rat ASC treatment. Injected rat ASCs co-existed with mesothelial cells in the sub-peritoneal layer. In vitro assays showed increased cellular proliferation of rat mesothelial cells combined with rat ASCs by co-culture assays, confirming that fluid factors from rat ASCs might play some role in facilitating the recovery of rat mesothelial cells. Hepatocyte growth factor was released from rat ASCs, and administration of recombinant hepatocyte growth factor increased rat mesothelial cell proliferation.ConclusionsBecause the peritoneal mesothelium shows strong expression of membrane complement regulators such as Crry, CD55 and CD59, restoration of the mesothelial cell layer by rat ASCs might prevent deposition of complement activation products and ameliorate peritoneal injuries. This study suggests the therapeutic possibilities of intraperitoneal rat ASC injection to suppress peritoneal inflammation by restoring the mesothelial layer and decreasing complement activation in fungal or yeast peritonitis.     

Analysis of possible factors relating to prognosis in autologous peripheral blood mononuclear cell transplantation for critical limb ischemia

Background aimsAutologous transplantation of granulocyte colony-stimulating factor–mobilized peripheral blood mononuclear cells (M-PBMNCs) has been shown to be effective in treating critical limb ischemia (CLI); however, the studies of the possible prognosis predictors after autologous M-PBMNC transplantation are inadequate. The objective of the study was to assess the possible factors affecting the results of M-PBMNC transplantation for CLI.MethodsWe reviewed the clinical profiles of 87 patients with CLI who were treated with M-PBMNC implantation in the Blood Diseases Hospital, Chinese Academy of Medical Sciences, between December 2002 and December 2011, and we followed these patients. The patients were divided into a good prognosis group and a poor prognosis group on the basis of whether amputation was performed. The significant differences of clinical variables between two groups were analyzed by means of the Mann-Whitney test and χ² test, and logistic regression analysis was used to study the variables representing the possible prognostic factors for amputation.ResultsOf the 87 patients, three patients died and one patient was lost during the follow-up period. We analyzed 83 patients. The diseases included CLI complicated by diabetes mellitus gangrene (35 cases, 42.2%), arteriosclerosis obliterans (31 cases, 37.3%) and thromboangiitis (17 cases, 20.5%). The mean age was 62 years (range, 30–87). The median follow-up time for the surviving patients was 5 years. The 5-year amputation-free rate was 72.2%, and no adverse effects related to M-PBMNC transplantation were observed.ConclusionsThe significant prognostic factors associated with poor angiogenesis were fibrinogen >4 g/L and fasting blood glucose >6 mmol/L.     

Culture expansion induces non-tumorigenic aneuploidy in adipose tissue-derived mesenchymal stromal cells

Background aimsAdipose tissue-derived mesenchymal stromal cells (ASCs) are of interest as a cell therapeutic agent for immunologic and degenerative diseases. During in vitro expansion, ASCs may be at risk for genetic alterations, and genetic screening is a prerequisite. We examined the presence of aneuploidy in ASCs and its origin and development during culture and evaluated the implications of aneuploidy for therapeutic use of ASCs.MethodsAdipose tissue of healthy individuals was used for isolation and expansion of ASCs. Chromosome copy numbers were studied using fluorescence in situ hybridization analysis. Aneuploidy was studied in freshly isolated ASCs, in ASCs cultured for 0–16 passages and in senescent cultures. To evaluate the plasticity of ploidy, ASCs were cloned, and the variation of ploidy in the clones was examined. Tumorigenicity was studied by subcutaneous injection of aneuploid ASCs in immunodeficient NOD/SCID mice.ResultsNo aneuploidy was detected in freshly isolated ASCs. In low passages (passages 0–4), aneuploidy was detected in 3.4% of ASCs. Prolonged culture expansion of ASCs (passages 5–16) resulted in a significant increase of aneuploidy to 7.1%. With senescence, aneuploidy increased further to 19.8%. Aneuploidy was observed in clones of diploid ASCs, demonstrating the de novo development of aneuploidy. No transformation of ASCs was observed, and in contrast to cancer cell lines, aneuploid ASCs were incapable of tumor formation in immunodeficient mice.ConclusionsASC cultures contain a stable percentage of aneuploid cells. Aneuploidy was not a predecessor of transformation or tumor formation. This finding indicates that aneuploidy is culture-induced but unlikely to compromise clinical application of ASCs.     

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells

BACKGROUNDAdipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.AIMTo test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).METHODSASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco''s Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).RESULTSDepending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSIONThe presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.     

Rat Adipose Tissue-Derived Stem Cells Transplantation Attenuates Cardiac Dysfunction Post Infarction and Biopolymers Enhance Cell Retention

Background

Cardiac cell transplantation is compromised by low cell retention and poor graft viability. Here, the effects of co-injecting adipose tissue-derived stem cells (ASCs) with biopolymers on cell cardiac retention, ventricular morphometry and performance were evaluated in a rat model of myocardial infarction (MI).

Methodology/Principal Findings

^99mTc-labeled ASCs (1×10⁶ cells) isolated from isogenic Lewis rats were injected 24 hours post-MI using fibrin a, collagen (ASC/C), or culture medium (ASC/M) as vehicle, and cell body distribution was assessed 24 hours later by γ-emission counting of harvested organs. ASC/F and ASC/C groups retained significantly more cells in the myocardium than ASC/M (13.8±2.0 and 26.8±2.4% vs. 4.8±0.7%, respectively). Then, morphometric and direct cardiac functional parameters were evaluated 4 weeks post-MI cell injection. Left ventricle (LV) perimeter and percentage of interstitial collagen in the spare myocardium were significantly attenuated in all ASC-treated groups compared to the non-treated (NT) and control groups (culture medium, fibrin, or collagen alone). Direct hemodynamic assessment under pharmacological stress showed that stroke volume (SV) and left ventricle end-diastolic pressure were preserved in ASC-treated groups regardless of the vehicle used to deliver ASCs. Stroke work (SW), a global index of cardiac function, improved in ASC/M while it normalized when biopolymers were co-injected with ASCs. A positive correlation was observed between cardiac ASCs retention and preservation of SV and improvement in SW post-MI under hemodynamic stress.

Conclusions

We provided direct evidence that intramyocardial injection of ASCs mitigates the negative cardiac remodeling and preserves ventricular function post-MI in rats and these beneficial effects can be further enhanced by administrating co-injection of ASCs with biopolymers.     

Application of autologous bone marrow mononuclear cells in six patients with advanced chronic critical limb ischemia as a result of diabetes: our experience

Background aimsPrevious clinical studies have reported that the injection of bone marrow (BM)-derived mononuclear cells (MNC) results in improvement in symptoms and healing of ulcers in patients with critical limb ischemia (CLI) up to stage IV of Fontaine's classification. However, most patients with Fontaine stage IV CLI limbs had to undergo amputation even after stem cell therapy. We report on six patients, who had poorly controlled diabetes with extensive ulceration and gangrene of limbs because of Fontaine stage IV CLI and had been advised amputation elsewhere, who underwent injection of autologous BM MNC.MethodsIn all six patients, BM was aspirated and the isolated MNC from the BM were injected intralesionally at various sites of the ulcer and its surroundings after necessary debridement. The patients were followed up at regular intervals for at least 6 months.ResultsAt the end of the 6-month follow-up, the lower limb pain and ulcers had improved significantly in all patients. The mean toe–brachial index had increased from 0.26 to 0.36. One patient died a month after therapy because of causes unrelated to the procedure. Limb salvage was possible in the remaining five patients and they had a pain-free walking distance of 100 m within 6 months.ConclusionsLimb salvage was possible in all six diabetic patients with Fontaine stage IV CLI following autologous BM MNC injection. The procedure was safe without any adverse outcomes.     

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro

Background aimsAdipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined.MethodsAfter isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test.ResultsDuring in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected.ConclusionsThe study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell–based therapy.     

Sitagliptin therapy enhances the number of circulating angiogenic cells and angiogenesis—evaluations in vitro and in the rat critical limb ischemia model

Background aimsWe tested the hypothesis that sitagliptin is capable of increasing blood flow in the rat critical limb ischemia (CLI) model by enhancement of angiogenesis.MethodsAdipose tissue from adult-male Fischer 344 rats (n = 6) were cultured in endothelial progenitor cell culture medium for 14 d with (25 μmol/L) or without sitagliptin. CLI was induced by ligation of the left femoral artery. Rats (n = 32) were equally separated into four groups: untreated controls (group 1), sitagliptin (4 mg/kg per day; group 2), CLI (group 3) and CLI with sitagliptin (group 4).ResultsIn vitro, 7 and 14 d after cell culture, endothelial progenitor cell biomarkers assessed by flow cytometry (Sca-1/CD31+, CXCR4+, c-kit+ and CD34+ cells) and Western blot (vascular endothelial growth factor, CXCR4 and stromal-derived factor [SDF]-1α) were remarkably higher in group 4 than in the other groups (all P < 0.01). In vivo, 2 and 14 d after the CLI procedure, circulating angiogenic cell (Sca-1/CD31+, Sca-1+ and CD31+) numbers were significantly higher in group 4 than in the other groups (all P < 0.001). Additionally, the messenger RNA and protein expression of angiogenic biomarkers (CXCR4, SDF-1α and vascular endothelial growth factor), immunofluorescent staining of angiogenic cells (CXCR4+, SDF-1α+, CD31+, von Willebrand factor + cells) and immunohistochemical staining of small vessel numbers in the ischemic area were significantly higher in group 4 than in the other groups (all P < 0.01). Furthermore, laser Doppler showed that the ratio of ischemic/normal blood flow was remarkably higher group 4 than in group 3 by days 14 and 28 after the CLI procedure (all P < 0.01).ConclusionsSitagliptin therapy enhances circulating angiogenic cell numbers, angiogenesis and blood flow in the CLI area.     

Enrichment isolation of adipose-derived stem/stromal cells from the liquid portion of liposuction aspirates with the use of an adherent column

Background aimsAdipose-derived stem/progenitor cells (ASCs) are typically obtained from the lipoaspirates; however, a smaller number of ASCs can be isolated without enzymatic digestion from the infranatant liposuction aspirate fluid (LAF). We evaluated the effectiveness of an adherent column, currently used to isolate mesenchymal stromal cells from bone marrow, to isolate LAF cells.MethodsWe applied peripheral blood (PB), PB mixed with cultured ASCs (PB-ASC), and LAF solution to the column and divided it into two fractions, the adherent (positive) and the non-adherent (negative) fractions. We compared this method with hypotonic hemolysis (lysis) for the red blood cell count, nucleated cells count and cell compositions as well as functional properties of isolated mesenchymal cells.ResultsThe column effectively removed red blood cells, though the removal efficiency was slightly inferior to hemolysis. After column processing of PB-ASC, 60.5% of ASCs (53.2% by lysis) were selectively collected in the positive fraction, and the negative fraction contained almost no ASCs. After processing of LAF solution, nucleated cell yields were comparable between the column and hemolysis; however, subsequent adherent culture indicated that a higher average ASC yield was obtained from the column-positive samples than from the lysis samples, suggesting that the column method may be superior to hemolysis for obtaining viable ASCs. Mesenchymal differentiation and network formation assays showed no statistical differences in ASC functions between the lysis and column-positive samples.ConclusionsOur results suggest that a column with non-woven rayon and polyethylene fabrics is useful for isolating stromal vascular fraction cells from LAF solutions for clinical applications.