Cryopreservation of adipose-derived stromal/stem cells using 1–2% Me2SO (DMSO) in combination with pentaisomaltose: An effective and less toxic alternative to comparable freezing media

Mesenchymal stromal/stem cells (MSCs) derived from bone marrow, umbilical cord and especially adipose tissue are increasingly being explored for their therapeutic potential to treat a wide variety of diseases. A prerequisite for most allogeneic off-the-shelf and some autologous MSC therapies is the ability to safely and efficiently cryopreserve cells during production or for storage prior to treatment. Dimethyl sulfoxide (Me₂SO) is still the commonly used gold standard cryoprotectant (CPA). However, undesirable cellular impacts and side effects of Me₂SO have led to an increasing demand for the development of safe and effective alternatives.This study investigated the effect of pentaisomaltose as a CPA for cryopreservation of adipose-derived stromal/stem cells (ASCs). We compared pentaisomaltose-based freezing media containing 1% Me₂SO (PIM1) or 2% Me₂SO (PIM2) to our in-house freezing media formulation containing 10% Me₂SO (STD10) and to CryoStor freezing media containing 2% or 10% Me₂SO (CS2 and CS10). We assessed the recovery of viable ASCs, their phenotype, differentiation potential, proliferation potential, and migratory potential. Further, their immunomodulatory potential was assessed by measuring their ability to suppress T cell proliferation and express immunomodulatory markers.The results showed that the post-thaw viability of ASCs cryopreserved with STD10, CS10 and PIM2 was improved compared to that of CS2. The recovery of ASCs with PIM1 and PIM2 was also improved compared to that of CS2. Proliferation and migration were comparable among the tested freezing media. The results showed no difference in the induction of PDL1, PDL2 or IDO1 expression. Nevertheless, the potential of cryopreserved ASCs to suppress T cell proliferation was reduced when the Me₂SO concentration was reduced (CS10>STD10>CS2 and PIM2>PIM1).Altogether, the migratory and immunomodulatory potential combined with improved recovery indicate that the addition of pentaisomaltose in the freezing media may allow for the reduction of the Me₂SO concentration to 2% while retaining a more potent cell product that what is recovered using comparable freezing media. With the desire to reduce the amount of Me₂SO, these results suggest that 2% and potentially even 1% Me₂SO in combination with 10% pentaisomaltose could be an effective and less toxic alternative to comparable freezing media.     

Therapeutic applications of mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. They have been demonstrated to play a role in tissue repair and regeneration in both preclinical and clinical studies. They also have remarkable immunosuppressive properties. We describe their application in settings that include the cardiovascular, central nervous, gastrointestinal, renal, orthopaedic and haematopoietic systems. Manufacturing of MSC for clinical trials is also discussed. Since tissue matching between MSC donor and recipient does not appear to be required, MSC may be the first cell type able to be used as an "off-the-shelf" therapeutic product.     

人间充质干细胞治疗系统性硬化症的研究进展

系统性硬化症(SSc)是一种罕见且累及全身多脏器的自身免疫性疾病。其病因和发病机制复杂,目前多以对症治疗为主。随着对干细胞研究的不断探索,干细胞治疗在医学领域的价值日益突显。越来越多的证据表明干细胞作为一种具有强大分化及再生潜能的有效手段在SSc患者的治疗中逐渐呈现出良好的效果。本文主要对3种常见来源的人间充质干细胞(hMSCs)包括骨髓hMSCs、脂肪hMSCs和脐带hMSCs在SSc患者中治疗的研究进展进行比较及展望,旨在为SSc患者的治疗提供更多依据。     

脂肪来源干细胞体外成骨和成脂及成神经的诱导分化研究

目的:探讨脂肪来源干细胞体外成骨和成脂及成神经的诱导分化情况。方法:选取10只SPF级雄性SD大鼠,将其不同部位的脂肪组织取出,分别采用不同方法对其向成骨、成脂及成神经等方向进行诱导分化并对其结果进行鉴定。结果:ADSC表达中,CD29占(99.11±0.13)%,CD44占(95.94±0.71)%,CD45占(0.12±0.09)%。经4周的成骨诱导后,茜素红S染色在细胞团中央发现红色钙化结节存在,碱性磷酸酶染色在细胞的胞质内观察到紫红色颗粒,经7d成脂诱导后,油红\"O\"染色在细胞质内观察到橙红色脂滴;经过6d的神经干培养基诱导后,通过免疫荧光染色证明诱导的Nestin细胞、神经丝蛋白-200以及GFAP等均出现阳性表达。结论:ADSC具备向脂肪、成骨及神经元等细胞进行多向分化的潜能,具有来源广、易于操作、体外增殖快速等优越性,并且不存在免疫排斥及医学伦理学问题,发展前景广阔。     

Ischemic flap survival improvement by composition-selective fat grafting with novel adipose tissue derived product - stromal vascular fraction gel

Background

Flap necrosis due to insufficient blood supply is a common postoperative complication in random pattern flaps. Stem cell therapies have emerged as promising biologics for tissue ischemia. A novel fat derived product, stromal vascular fraction gel (SVF-gel), can be prepared with lipoaspirate through simple mechanical processing, removing only the lipid content. SVF-gel enriches adipose-derived stem cells and potentially beneficial for flap necrosis.

Regulatory T cells (CD4CD25FoxP3) expansion in systemic sclerosis correlates with disease activity and severity

Background

The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc).

Methods

Ten patients with SSc donated 20 ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4⁺ cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-β and IL-10 by activated CD4⁺ cells was measured by ELISA in culture supernatants.

Results

The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r = 0.71, p = 0.034 and r = 0.67, p = 0.044, respectively). ELISA-measured production of TGF-β and IL-10 by CD4⁺ cells was similar in patients and controls.

Conclusions

Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-β or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.     

PKC-delta induces cardiomyogenic gene expression in human adipose-derived stem cells

Stromal cells from fat tissues exhibit properties of mesenchymal stem cells from other sources with the ability to differentiate towards multiple cell types. However, effective differentiation of these mesenchymal cells, called adipose-derived stem cells (ADSCs), towards cardiomyogenic lineage has been limited to a small number of isolated clones in an extended culture. Previously, we reported that treatment with phorbol ester induces the expression of several cardiomyogenic genes in the absence of serum. This study was performed to identify the roles of PKC isoforms in cardiomyogenic gene expression of ADSCs. Treatment with 10 nM phorbol myristate acetate (PMA) for 24 h caused sustained increases in mRNA levels for various cardiomyogenic genes, such as Mef2C, cardiac actin and troponin, for at least 6 days following the drug removal. The use of various inhibitors specific for PKC isoforms demonstrated that the novel PKC-theta/delta isoforms mediate the PMA effects. RT-PCR revealed that ADSCs express significant mRNA for PKC-delta, but not theta isoform. Overexpression of cDNA for PKC-delta resulted in marked increases in cardiac mRNA expression. These results indicate that activation of PKC-delta induces the expression of multiple cardiomyogenic genes in ADSCs.     

mitoTEMPO改善糖尿病小鼠脂肪干细胞的氧化损伤

目的:探讨线粒体靶向抗氧化剂mitoTEMPO对糖尿病小鼠脂肪干细胞(Adipose-derived stem cells,ADSCs)氧化损伤的影响。方法:采用60%高脂饮食喂养雄性C57小鼠(4周龄)连续8周,并在高脂喂养第2周,对小鼠进行连续5天腹腔注射低剂量链脲佐菌素(streptozotocin,STZ)(25 mg·kg-1)制备2型糖尿病小鼠模型。喂养2周后,检测小鼠血浆葡萄糖水平等指标符合2型糖尿病标准者纳入实验。分别从正常小鼠与STZ诱导的糖尿病小鼠的腹股沟处皮下脂肪组织分离培养脂肪干细胞(ADSCs),并将其各分为4组:DMEM培养的正常ADSCs组(nADSCs组),DMEM培养的糖尿病ADSCs组(dADSCs组),TEMPO治疗的糖尿病ADSCs组(T-dADSCs组),mitoTEMPO治疗的糖尿病ADSCs组(mitoT-dADSCs组)。采用细胞计数试剂盒-8(CCK-8)检测细胞存活能力;油红-O和茜素红染色分别检测成脂细胞分化与成骨细胞分化能力;划痕实验和Transwell试验分别测定细胞迁移能力;DCF和mito SOX染色荧光成像分别检测细胞内和线粒体中的活性氧簇(Reactive oxygen species, ROS)水平。结果:①与nADSCs组相比,d ADSCs组的细胞活力明显下降(P0.05)、成骨细胞分化与成脂细胞分化程度明显下降(P0.05)、脂肪干细胞迁移能力明显下降(P0.05)、细胞内和线粒体中ROS水平明显升高(P0.05)。②与dADSCs组相比,T-dADSCs和mitoT-dADSCs组的细胞内和线粒体中的ROS水平明显降低(P0.05);与dADSCs组相比,mitoT-dADSCs组的成骨细胞分化与成脂细胞分化能力明显提升(P0.05),基本达到nADSCs组的分化水平;与dADSCs组相比,mitoT-dADSCs治疗组的细胞迁移能力显著升高(P0.05)、T-dADSCs组的细胞迁移能力增长无明显差异。结论:mitoTEMPO可以减轻糖尿病时线粒体内活性氧簇蓄积造成的脂肪干细胞的氧化应激损伤与功能紊乱。