The potential of adipose‐derived stem cells in craniofacial repair and regeneration

The recent identification of a mesenchymal stem cell population in adipose tissue has led to an abundance of research focused on the regenerative properties of these cells. As such, adipose‐derived stem cells (ASCs) and potential therapies in craniofacial regeneration have been widely studied. This review will discuss the identification and potential of ASCs, and specifically, preclinical and clinical studies using ASCs in craniofacial repair. Studies involving ASCs in the repair of defects caused by craniosynostosis and Treacher Collins syndrome will be discussed. A comprehensive review of the literature will be presented, focusing on fat grafting and biomaterials‐based approaches that include ASCs for craniofacial regeneration. (Part C) 96:95–97, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

一种体外快速分离脂肪来源干细胞的方法

目的:以小鼠为模型,建立一种基于流式细胞仪为检测手段的快速分离脂肪来源干细胞的方法,解决间充质干细胞进入实际应用过程中遇到的难题。方法:取BALB/c小鼠腹股沟内侧的皮下脂肪组织,采用Ⅰ型胶原酶消化等系列措施,获取脂肪干细胞(adipose-derived stem cells,ADSCs)。所得细胞分离培养4代后,流式细胞仪分选出CD73+CD45-ADSCs后成骨诱导分化。碱性磷酸酶染色和实时荧光定量PCR检测其分化情况。结果:刚分离培养的ADSCs细胞普遍呈圆形或椭圆形,传至第三代的细胞,非MSCs细胞逐渐被淘汰,剩余的细胞形态逐渐变得一致,细胞形态呈梭形。流式细胞仪检测发现ADSCs细胞表面抗原标记CD73+CD45-为20.7%。所得的ADSCs成骨诱导分化后碱性磷酸酶(alkaline phosphatase,ALP)染色呈阳性,实时荧光定量PCR检测成骨标志基因发现它们表达上调,其中ALP的表达高达22倍。结论:本方法可以获得纯度较高的ADSCs,且耗时少成本低;且提示可采用该方法来获得大量的人源ADSCs用于组织工程修复。     

VEGF receptor Flk-1 plays an important role in c-kit expression in adipose tissue derived stem cells

It is known that c-kit(+) cells are increased in heart after infarction. The exact origins of the cardiac c-kit(+) cells remain to be determined. We asked whether adipose tissue could be a potential source of c-kit(+) cells. Our data show that the number of c-kit(+) cells increased in adipose tissue derived stem cells when cultured with conditioned medium from neonatal cardiomyocytes grown under serum deprivation and hypoxia condition. We also found that VEGF receptor Flk-1 is involved in c-kit up regulation via ERK-mediated pathway.     

Monolayer cell expansion conditions affect the chondrogenic potential of adipose-derived stem cells

Adipose-derived stem cells (ASCs) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue. Isolated ASCs are typically expanded in monolayer on standard tissue culture plastic with a basal medium containing 10% fetal bovine serum. However, recent data suggest that altering the monolayer expansion conditions by using suspension culture plastic, adding growth factors to the medium, or adjusting the seeding density may affect the self-renewal rate, multipotency, and lineage-specific differentiation potential of the ASCs. We hypothesized that variation in any of these expansion conditions would influence the chondrogenic potential of ASCs. ASCs were isolated from human liposuction waste tissue and expanded through two passages with different tissue culture plastic, feed medium, and cell seeding densities. Once expanded, the cells were cast in an agarose gel and subjected to identical chondrogenic culture conditions for 7 days, at which point cell viability, radiolabel incorporation, and gene expression were measured. High rates of matrix synthesis upon chondrogenic induction were mostly associated with smaller cells, as indicated by cell width and area on tissue culture plastic, and it appears that expansion in a growth factor supplemented medium is important in maintaining this morphology. All end-point measures were highly dependent on the specific monolayer culture conditions. These results support the hypothesis that monolayer culture conditions may "prime" the cells or predispose them towards a specific phenotype and thus underscore the importance of early culture conditions in determining the growth and differentiation potential of ASCs.     

Adipose-derived stromal cells: Their identity and uses in clinical trials, an update

In adults, adipose tissue is abundant and can be easily sampled using liposuction. Largely involved in obesity and associated metabolic disorders, it is now described as a reservoir of immature stromal cells. These cells, called adipose-derived stromal cells (ADSCs) must be distinguished from the crude stromal vascular fraction (SVF) obtained after digestion of adipose tissue. ADSCs share many features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but they also display some specific features, including a greater angiogenic potential. Their angiogenic properties as well as their paracrine activity suggest a putative tumor-promoting role for ADSCs although contradictory data have been published on this issue. Both SVF cells and ADSCs are currently being investigated in clinical trials in several fields (chronic inflammation, ischemic diseases, etc. ). Apart from a phase Ⅲ trial on the treatment of fistula,most of these are in phaseⅠand use autologous cells. In the near future, the end results of these trials should provide a great deal of data on the safety of ADSC use.     

Characterization and comparison of adipose tissue‐derived cells from human subcutaneous and omental adipose tissues

Different fat depots contribute differently to disease and function. These differences may be due to the regional variation in cell types and inherent properties of fat cell progenitors. To address the differences of cell types in the adipose tissue from different depots, the phenotypes of freshly isolated adipose tissue‐derived cells (ATDCs) from subcutaneous (SC) and omental (OM) adipose tissues were compared using flow cytometry. Our results showed that CD31^?CD34⁺CD45^?CD90^‐CD105^?CD146⁺ population, containing vascular smooth muscle cells and pericytes, was specifically defined in the SC adipose tissue while no such population was observed in OM adipose tissue. On the other hand, CD31^?CD34⁺CD45^?CD90^?CD105^?CD146^? population, which is an undefined cell population, were found solely in OM adipose tissue. Overall, the SC adipose tissue contained more ATDCs than OM adipose tissue, while OM adipose tissue contained more blood‐derived cells. Regarding to the inherent properties of fat cell progenitors from the two depots, adipose‐derived stem cells (ADSCs) from SC had higher capacity to differentiate into both adipogenic and osteogenic lineages than those from OM, regardless of that the proliferation rates of ADSCs from both depots were similar. The higher differentiation capacity of ADSCs from SC adipose tissue suggests that SC tissue is more suitable cell source for regenerative medicine than OM adipose tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Adipose-derived stem cells decreased microglia activation and protected dopaminergic loss in rat lipopolysaccharide model

Adult stem cell therapy is being used extensively to rejuvenate damaged tissue. One important tissue source to obtain these cells is adipose, which contains cells called adipose-derived stem cells (ADSCs). These cells have a great therapeutic potential not only for their multipotent properties as well as for immunomodulatory effects on the immune system. Parkinson's disease is characterized as neurodegenerative disorder which etiology is undoubtedly related to neuroinflammation process. The properties of ADSCs can be used as a new tool in stem cells therapy to treat neurodegenerative disorders. However, their efficacies are still controversial. Some authors have reported neuroprotection effects, while others did not find differences or stem cells increased the damage. Our previous study showed that ADSCs can survive long time after transplantation, suggesting us some biological effects could need more time to be repaired. In this study, we assessed the neuroprotection 6 months after transplantation. Our results suggest ADSCs can protect the dopaminergic loss after lipopolysaccharide (LPS) injection both reducing the microglia activation and differentiating into dopaminergic cells.     

Combined pre‐conditioning with salidroside and hypoxia improves proliferation,migration and stress tolerance of adipose‐derived stem cells

Oxidative stress after ischaemia impairs the function of transplanted stem cells. Increasing evidence has suggested that either salidroside (SAL) or hypoxia regulates growth of stem cells. However, the role of SAL in regulating function of hypoxia‐pre–conditioned stem cells remains elusive. Thus, this study aimed to determine the effect of SAL and hypoxia pre‐conditionings on the proliferation, migration and tolerance against oxidative stress in rat adipose‐derived stem cells (rASCs). rASCs treated with SAL under normoxia (20% O₂) or hypoxia (5% O₂) were analysed for the cell viability, proliferation, migration and resistance against H₂O₂‐induced oxidative stress. In addition, the activation of Akt, Erk1/2, LC3, NF‐κB and apoptosis‐associated pathways was assayed by Western blot. The results showed that SAL and hypoxia treatments synergistically enhanced the viability (fold) and proliferation of rASCs under non‐stressed conditions in association with increased autophagic flux and activation of Akt, Erk1/2 and LC3. H₂O₂‐induced oxidative stress, cytotoxicity, apoptosis, autophagic cell death and NF‐κB activation were inhibited by SAL or hypoxia, and further attenuated by the combined SAL and hypoxia pre‐treatment. The SAL and hypoxia pre‐treatment also enhanced the proliferation and migration of rASCs under oxidative stress in association with Akt and Erk1/2 activation; however, the combined pre‐treatment exhibited a more profound enhancement in the migration than proliferation. Our data suggest that SAL combined with hypoxia pre‐conditioning may enhance the therapeutic capacity of ASCs in post‐ischaemic repair.     

Human adipose stromal cell therapy improves survival and reduces renal inflammation and capillary rarefaction in acute kidney injury

Damage to endothelial cells contributes to acute kidney injury (AKI) by causing impaired perfusion, while the permanent loss of the capillary network following AKI has been suggested to promote chronic kidney disease. Therefore, strategies to protect renal vasculature may impact both short‐term recovery and long‐term functional preservation post‐AKI. Human adipose stromal cells (hASCs) possess pro‐angiogenic and anti‐inflammatory properties and therefore have been tested as a therapeutic agent to treat ischaemic conditions. This study evaluated hASC potential to facilitate recovery from AKI with specific attention to capillary preservation and inflammation. Male Sprague Dawley rats were subjected to bilateral ischaemia/reperfusion and allowed to recover for either two or seven days. At the time of reperfusion, hASCs or vehicle was injected into the suprarenal abdominal aorta. hASC‐treated rats had significantly greater survival compared to vehicle‐treated rats (88.7% versus 69.3%). hASC treatment showed hastened recovery as demonstrated by lower creatinine levels at 48 hrs, while tubular damage was significantly reduced at 48 hrs. hASC treatment resulted in a significant decrease in total T cell and Th17 cell infiltration into injured kidneys at 2 days post‐AKI, but an increase in accumulation of regulatory T cells. By day 7, hASC‐treated rats showed significantly attenuated capillary rarefaction in the cortex (15% versus 5%) and outer medulla (36% versus 18%) compared to vehicle‐treated rats as well as reduced accumulation of interstitial alpha‐smooth muscle actin‐positive myofibroblasts. These results suggest for the first time that hASCs improve recovery from I/R‐induced injury by mechanisms that contribute to decrease in inflammation and preservation of peritubular capillaries.     

A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model.     

Human adipose derived stroma/stem cells grow in serum-free medium as floating spheres

With the goal of obtaining clinically safe human adipose-derived stroma/stem cells (ASC) and eliminating the use of serum, we have developed a new culture system that allows the expansion of ASC as spheres in a defined medium. These spheres can be passaged several times. They are not only aggregated cells but rather originate from single cells as clonal spheres can be obtained after seeding at very low density and reform clonal spheres after dissociation. These spheres can also revert to monolayer growth when plated in medium containing human plasma and even generate fibroblast-like colonies (CFU-f). Under several differentiation-specific media, spheres-derived ASC maintain their capacity to differentiate into osteoblasts, endothelial cells and adipocytes. These results indicate that human ASC can be maintained in a serum-free 3D culture system, which is of great interest for the expansion in bioreactors of autologous ASC and their use in clinical trials.     

谷氨酸对大鼠脂肪干细胞诱导分化神经细胞的影响及机制

目的:研究谷氨酸(Glu)对大鼠脂肪干细胞Adipose-derived stem cells(ADCSs)诱导分化神经细胞的作用及机制。方法:取成年大鼠腹股沟脂肪组织进行体外细胞培养,采用免疫组化方法检测证实为ADSCs。对照组为正常培养的ADSCs并诱导分化神经细胞,谷氨酸(Glu)处理组加入不同浓度的Glu,MTT比色法观察脂肪干细胞的存活率。结果:从ADSCs诱导分化的细胞包括神经元及神经胶质细胞,免疫组化结果显示神经元特异性烯醇化酶(NSE)染色阳性和胶质纤维酸性蛋白(GFAP)染色阳性。Glu处理组给药24h后,与对照组比较ADSCs存活率明显降低,50μmol·L-1Glu组细胞存活率为83.98%,与对照组比较差异无显著性(P>0.05);100及1000μmol·L-1Glu组干细胞存活率(分别为66.82%和17.08%)低于对照组(P<0.05,P<0.01)。结论:Glu对ADSCs有损伤作用,随着Glu剂量的增加,ADSCs的存活率逐渐降低,二者呈剂量依赖关系。     

Intra‐articular injection of autologous adipose‐derived mesenchymal stem cells in the treatment of knee osteoarthritis

Background

Osteoarthritis (OA) is a chronic degenerative joint disease and is considered to be the fourth leading cause of disability and the second cause of inability to work in men. Recently, adipose‐derived mesenchymal stem cells (AD‐MSCs) came into focus for regenerative medicine as a promising tool for the treatment of OA. The administration of stem cells into impaired joints results in pain relief and improves quality of life, accompanied by restoration of hyaline articular cartilage.

Methods

In the present study, nine patients (including two patients with bilateral symptoms) diagnosed with osteoarthritis (International Knee Documentation grade B in 5 and grade D in six knees) were treated using a single injection of AD‐MSCs at a concentration of 0.5–1.0 × 10⁷ cells and were followed up for 18 months. During follow‐up, all the cases were evaluated clinically by Knee Society score (KSS), Hospital for Special Surgery knee score (HSS‐KS), Tegner–Lysholm (T–L) score and visual analogue scale (VAS) of pain, as well as by plain radiography and by magnetic resonance imaging visualization with 2D Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score assessment.

Results

Significant improvement of all four clinical scores was observed within the first 6 months (KSS for 41.4 points, HSS‐KS for 33.9 points, T–L score for 44.8 points, VAS of pain from 54.5 to 9.3) and improvement persisted throughout the rest of the follow‐up. MOCART score showed significant cartilage restoration (from 43 ± 7.2 to 63 ± 17.1), whereas radiography showed neither improvement, nor further joint degeneration.

Conclusions

The results obtained in the present study provide good basis for prospective randomized controlled clinical trials with respect to the use of AD‐MSCs in the treatment of osteoarthritis.     

Therapeutic role of adipose tissue–derived stem cells versus microvesicles in a rat model of cerebellar injury

Monosodium glutamate (MSG) is a controversial food additive reported to cause negative effects on public health. Adipose stem cells (ASCs) and their derived vesicles (MVs) represent a promising cure for human diseases. This work was planned to compare the therapeutic effects of adipose stem cells and microvesicles in MSG‐induced cerebellar damage. Forty adult healthy male Wister rats were equally divided into four groups: Group I (control group), group II (MSG‐treated), group III (MSG/ASCs‐treated), and group IV (MSG/MVs‐treated). Motor behaviour of rats was assessed. Characterization of ASCs and MVs was done by flow cytometry. The cerebellum was processed for light and electron microscopic studies, and immunohistochemical localization of PCNA and GFAP. Morphometry was done for the number of Purkinje cells in H&E‐stained sections, area per cent of GFAP immune reactivity and number of positive PCNA cells. Our results showed MSG‐induced deterioration in the motor part. Moreover, MSG increases oxidant and apoptotic with decreases of antioxidant biomarkers. Structural changes in the cerebellar cortex as degeneration of nerve cells and gliosis were detected. There were also a decrease in the number of Purkinje cells, an increase in the area per cent of GFAP immune reactivity and a decrease in the number of positive PCNA cells, as compared to the control. Rats treated with ASCs showed marked functional and structural improvement in comparison with MV‐treated rats. Thus, both ASCs and MVs had therapeutic potential for MSG‐induced cerebellar damage with better results in case of ASCs.