Venugopal Parvathy, Balasubramanian Sudha, Majumdar Anish Sen, Ta Malancha
Stempeutics research Pvt. Ltd, Manipal Hospital, Bangalore, India.
Stem Cells Cloning. 2011 Apr 21;4:39-50. doi: 10.2147/SCCAA.S17548. eCollection 2011.
Mesenchymal stem cells (MSCs) have become an attractive tool for tissue engineering and targets in clinical transplantation due to their regeneration potential and immuno-suppressive capacity. Although MSCs derived from bone marrow are the most widely used, their harvest requires an invasive procedure. The umbilical cord, which is discarded at birth, can provide an inexhaustible source of stem cells for therapy. The Wharton's jelly-derived MSCs (WJ-MSCs), from the umbilical cord, have been shown to have faster proliferation rates and greater expansion capability compared with adult MSCs. The standard isolation and in vitro culture protocol for WJ-MSCs utilizes fetal bovine serum (FBS) or calf serum as a nutrient supplement. However, FBS raises potential safety concerns such as transmission of viral/prion disease and may initiate xenogeneic immune reactions against bovine antigens. Therefore, for therapeutic applications, there is an urgent requirement to establish an alternative nutrient supplement which would favor cell proliferation, retain MSC characteristics, and prove safe in human subjects. In the present study, we isolated and expanded WJ-MSCs in 5% pooled, allogeneic human serum (HS) supplemented with 2 ng/mL of basic fibroblast growth factor. For cell dissociation, porcine trypsin was replaced with TrypLE, a recombinant enzyme, and a protease-free protocol was adapted for isolation of MSCs from WJ. We determined their growth kinetics, in vitro differentiation potential, surface marker expression, and colony-forming unit potential and compared them against standard WJ-MSC cultures expanded in 10% FBS. All these parameters matched quite well between the two MSC populations. To test whether there is any alteration in gene expression on switching from FBS to HS, we analyzed a panel of stem cell and early lineage markers using Taqman® low density array. No significant deviation in gene expression was observed between the two populations. Thus we established an efficient, complete xeno-free protocol for propagation of human WJ-MSCs.
间充质干细胞（MSCs）因其再生潜力和免疫抑制能力，已成为组织工程和临床移植靶点的一种有吸引力的工具。尽管源自骨髓的MSCs是使用最广泛的，但获取它们需要侵入性操作。出生时被丢弃的脐带可为治疗提供无穷无尽的干细胞来源。已证明，源自脐带华通氏胶的MSCs（WJ-MSCs）与成人MSCs相比，具有更快的增殖速率和更强的扩增能力。WJ-MSCs的标准分离和体外培养方案使用胎牛血清（FBS）或小牛血清作为营养补充剂。然而，FBS引发了潜在的安全问题，如病毒/朊病毒疾病的传播，并可能引发针对牛抗原的异种免疫反应。因此，对于治疗应用，迫切需要建立一种替代营养补充剂，它有利于细胞增殖，保留MSCs特性，并在人体中证明是安全的。在本研究中，我们在补充有2 ng/mL碱性成纤维细胞生长因子的5%混合异体人血清（HS）中分离并扩增WJ-MSCs。对于细胞解离，用重组酶TrypLE替代猪胰蛋白酶，并采用无蛋白酶方案从WJ中分离MSCs。我们测定了它们的生长动力学、体外分化潜力、表面标志物表达和集落形成单位潜力，并将它们与在10% FBS中扩增的标准WJ-MSC培养物进行比较。这两个MSC群体的所有这些参数都相当匹配。为了测试从FBS转换到HS时基因表达是否有任何变化，我们使用Taqman®低密度阵列分析了一组干细胞和早期谱系标志物。在这两个群体之间未观察到基因表达的显著偏差。因此，我们建立了一种高效、完全无动物源的人WJ-MSCs扩增方案。
