Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Exosomes derived from adipose tissue,bone marrow,and umbilical cord blood for cardioprotection after myocardial infarction

Human mesenchymal stem cells (MSCs) have the potential for improving cardiac function following myocardial infarction (MI). This study was performed to explore the cardioprotection of bone marrow mesenchymal stem cells (BMMSCs), adipose tissue-derived mesenchymal stem cells (ADMSCs), and umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) for myocardium in rats after MI. MI models were established in rats, which were injected with PBS, BMMSCs, ADMSCs, and UCMSCs. Cardiac function was detected by ultrasonic cardiogram. TTC staining, TUNEL staining, and immunohistochemistry were adopted to determine infarction area, cardiomyocyte apoptosis, and microvascular density (MVD), respectively. Exosomes were derived from BMMSCs, ADMSCs, and UCBMSCs, and identified by morphological observation and CD63 expression detection. Neonatal rat cardiomyocytes (NRCMs) were isolated and cultured with hypoxia, subjected to PBS and exosomes derived from BMMSCs, ADMSCs, and UCMSCs. Flow cytometry and enzyme-linked immunosorbent assay were used to determine NRCM apoptosis and the levels of angiogenesis-related markers (VEGF, bFGF, and HGF). According to ultrasonic cardiogram, BMMSCs, ADMSCs, and UCMSCs facilitated the cardiac function of MI rats. Furthermore, three kinds of MSCs inhibited cardiomyocyte apoptosis, infarction area, and increased MVD. NRCMs treated with exosomes derived from BMMSCs, ADMSCs, and UCMSCs reduced the NRCM apoptosis and promoted angiogenesis by increasing levels of VEGF, bFGF, and HGF. Notably, exosomes from ADMSCs had the most significant effect. On the basis of the results obtained from this study, exosomes derived from BMMSCs, ADMSCs, and UCBMSCs inhibited the cardiomyocyte apoptosis and promoted angiogenesis, thereby improving cardiac function and protecting myocardium. Notably, exosomes from ADMSCs stimulated most of the cardioprotection factors.     

人羊膜间充质细胞具有向心肌样细胞分化的特性

摘 要 探讨人羊膜间充质细胞（human amniotic mesenchymal cells,hAMCs）向心肌细胞分化的能力。采用胶原酶消化法分离hAMCs,用流式细胞仪进行表型鉴定;用5-氮杂胞苷和碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）诱导hAMCs向心肌细胞分化,免疫荧光染色法检测诱导后细胞中特异蛋白结蛋白和α-辅肌动蛋白的表达,RT-PCR检测心肌特异性转录因子Nkx2.5 、GATA-4和心肌特异性收缩蛋白α-肌球蛋白重链（α-myosin heavy chain,α-MHC）mRNA的表达。结果显示：①hAMCs原代培养至第6 d,贴壁细胞汇合度可达80%,呈漩涡状生长。传代后hAMCs增殖迅速,3~4 d细胞汇合度可达100%,细胞呈梭形或多角形。②hAMCs表达CD44和波形蛋白,不表达CK19。③hAMCs经诱导分化8~10 d后细胞排列紧密,多为长梭形。③hAMCs诱导2 w和4 w表达α-辅肌动蛋白和心肌特异性转录因子Nkx2.5。④诱导前后的hAMCs均表达结蛋白和GATA-4,但均未见α-MHC表达。说明hAMCs具有向心肌样细胞分化的能力,可望成为细胞心肌成形术（cellular cardiomyoplasty,CCM）的候选细胞。     

Prospect of mesenchymal stem cells in therapy of osteoporosis: A review

Osteoporosis is a systemic skeletal disease associated with reduced bone strong point that results in raised fracture risk, with decreased bone strength, leading to reduced bone mineral density and poor bone quality. It is the most common in older females but some men are also at high risk. Although considered as a predictable result of aging, it is can be avoidable and treatable. The existing treatment of osteoporosis mainly contains antiresorptive and anabolic agents. In spite of these improvements, concerns around unusual side-effects of antiresorptive drugs, and the lack of perfect confirmation in maintenance of their long-standing effectiveness is bring about many patients not receiving these drugs. Over the years, the stem cell-based therapy has attained substantial clinical consideration because of its potential to treat numerous diseases. The stem cell therapy has been recommended as a probable therapeutic approach for patients with osteoporosis. Even though the concept of stem cell-based therapy for osteoporosis has caught substantial attention, no clinical trial has been published on humans. The cell studies based on osteoporosis are primarily focused on osteoclastic activity and bone resorption procedures. Earlier, it was on osteoblastogenesis and in recent times, on the differentiation probable of mesenchymal stem cells. In this review, we have summarized the therapeutic role of stem cell-based strategy in osteoporosis.     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

内皮细胞向间充质细胞的转分化

内皮细胞向间充质细胞转分化(endothelial-to-mesenchymal transition,EndMT)过程中,内皮从内皮层分离,获得间充质细胞表型,其特征是细胞连接能力下降,内皮标志物减少,获得间充质细胞标志物,并具有扩散和迁移特性。在受损组织中,内皮细胞能够通过EndMT转分化为成纤维细胞,因此具有EndMT特性的内皮细胞在组织重构和纤维化中发挥重要作用。EndMT不仅参与心脏发育这一重要过程也参与许多病理性疾病如癌症,心脏的纤维化和肺动脉高压等。所以,靶向EndMT对多种疾病具有潜在的治疗意义。     

Mesenchymal stem cells induce dermal fibroblast responses to injury

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.     

Bioimaging for the monitoring of the in vivo distribution of infused mesenchymal stem cells in a mouse model of the graft-versus-host reaction

Cell therapy using MSCs (mesenchymal stem cells) might be effective treatment for refractory GVHD (graft-versus-host disease). However, the fate and distribution of MSCs after transplantation remains unclear. In this study, an animal model was developed to monitor the dynamic distribution of MSCs in mice with GVHD. A GVHD mouse model was established by transplanting C57BL/6 donor bone marrow cells and C57BL/6 EGFP (enhanced green fluorescent protein) splenocytes into lethally irradiated BALB/c nude recipient mice. Donor MSCs were obtained from MHC-identical C57BL/6 RFP (red fluorescent protein) mice and infused into the recipient mice on the same transplantation day. In vivo movement of the donor splenocytes (EGFP) and MSCs (RFP) were evaluated by measuring the biofluorescence (IVIS-Xenogen system). Donor splenocytes and MSCs reached the lungs first, and then the gastrointestinal tract, lymph nodes and skin, in that order; the transit time and localization site of these cells were very similar. In the recipient mouse with GVHD, the number of detectable cells declined with time, as assessed by biofluorescence imaging and confirmed by RT (real-time)-PCR. This bioimaging system might be useful for preclinical testing and the design of therapeutic strategies for monitoring the dynamic distribution of MSCs with GVHD.     

Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers

We first identified and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). Cells from osteoarthritic cartilage were enzymatically isolated and analyzed directly or after culture expansion over several passages by FACS using various combinations of surface markers that have been identified on human MPCs (CD9, CD44, CD54, CD90, CD166). Culture expanded cells combined and the subpopulation derived from initially sorted CD9+, CD90+, CD166+ cells were tested for their osteogenic, adipogenic and chondrogenic potential using established differentiation protocols. The differentiation was analyzed by immunohistochemistry and by RT-PCR for the expression of lineage related marker genes. Using FACS analysis we found that various triple combinations of CD9, CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2-12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints.     

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

The need for efficient and reliable technologies for clinical‐scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood‐derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10⁷ cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10⁸ cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra‐Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier‐based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical‐scale production system. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 568–572, 2013     

骨髓间充质干细胞免疫抑制作用及其机理

近年来间充质干细胞特殊的免疫学特性越来越引起人们的重视,使其成为移植领域的一个研究热点。许多实验室就间充质干细胞的免疫抑制作用和机理做了大量的研究工作,取得了一定的进展。本文简要综述间充质干细胞的免疫抑制作用和机理。     

骨与关节系统发生过程中相关调控机制的研究进展

脊椎动物胚胎期骨与关节系统的发生是一种复杂生命现象,起始于中胚层间充质细胞的定向聚集,形成肢芽,然后在一系列作用因子的调控下,肢芽内细胞进一步分化,形成具有骨骼雏形的软骨原基,后者经软骨内骨化发育成骨。四肢骨大多是以这种方式发生的,四肢的滑膜关节系统也随骨骼的发生而形成。详细阐述了近年来对肢体骨与关节系统发生各步骤相关调控机制方面的研究进展。Abstract: The embryonic development of bone and joint involves in complicated events for vertebrate limb. It originates from determined condensation of mesenchymal cells from lateral mesoderm. These cells and the overlying ectodermal jacket form limb buds at presumptive limb levels. Then, under the control of systemic factors, mesenchymal cells aggregate and differentiate to form catilage blastemal elements that prefigure skeletal limb components. The latter develops into skeleton through endochondral ossification. The majority of the bones of the limb form by the endochondral mechanism. The formation of synovial joint system and bone development occur simultaneously. This article reviewed the progress on the related control mechanism in the development of bone and joint recently.     

Safety and efficacy of allogenic placental mesenchymal stem cells for treating knee osteoarthritis: a pilot study

Objective

Knee osteoarthritis (OA) is a common skeletal impairment that can cause many limitations in normal life activities. Stem cell therapy has been studied for decades for its regenerative potency in various diseases. We investigated the safety and efficacy of intra-articular injection of placental mesenchymal stem cells (MSCs) in knee OA healing.

Mesenchymal stem cells: Paracrine signaling and differentiation during cutaneous wound repair

Cutaneous wounds persist as a health care crisis in spite of increased understanding of the cellular and molecular responses to injury. Contributing significantly to this crisis is the lack of reliable therapies for treatment of wounds that are slow to heal including chronic wounds and deep dermal wounds that develop hypertrophic scars. This article will review the growing evidence demonstrating the promise of multipotent mesenchymal stem/stromal (MSCs) for the treatment of impaired wound healing. MSCs are often referred to as mesenchymal stem cells despite concerns that these cells are not truly stem cells given the lack of evidence demonstrating self-renewal in vivo. Regardless, abundant evidence demonstrates the therapeutic potential of MSCs for repair and regeneration of damaged tissue due to injury or disease. To date, MSC treatment of acute and chronic wounds results in accelerated wound closure with increased epithelialization, granulation tissue formation and angiogenesis. Although there is evidence for MSC differentiation in the wound, most of the therapeutic effects are likely due to MSCs releasing soluble factors that regulate local cellular responses to cutaneous injury. Important challenges need to be overcome before MSCs can be used effectively to treat wounds that are slow to heal.     

Human adipose-derived mesenchymal stem cells: serial passaging,doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

间充质干细胞特性与应用前景

间充质干细胞是中胚层发育的早期细胞,具备干细胞的基本特性。在发育的不同阶段和特定环境条件下,间充质干细胞可向骨、软骨、肌肉、神经、血管及血液细胞等多种方向分化。在成体的很多器官和组织中也存在着间充质干细胞,以备修复和再生所用。间充质干细胞易于体外培养,扩增迅速,可以分化为多种细胞,为干细胞生物工程提供了一个很好的种子细胞。在明确间充质干细胞生物学特性和分化的机制后,可在体外和体内将其定向诱导分化为多种细胞。间充质干细胞具有巨大的临床应用价值和科学研究价值。