Department of Immunology, Institute for Cellular and Molecular Medicine, SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, PO Box 2034, Pretoria, 0001, South Africa.
Department of Oral Pathology and Oral Biology, School of Dentistry, Faculty of Health Sciences, University of Pretoria, PO Box 1266, Pretoria, 0001, South Africa.
Stem Cell Res Ther. 2019 Aug 14;10(1):253. doi: 10.1186/s13287-019-1370-2.
For adipose-derived stromal cells (ASCs) to be safe for use in the clinical setting, they need to be prepared using good manufacturing practices (GMPs). Fetal bovine serum (FBS), used to expand ASCs in vitro in some human clinical trials, runs the risk of xenoimmunization and zoonotic disease transmission. To ensure that GMP standards are maintained, pooled human platelet lysate (pHPL) has been used as an alternative to FBS. ASCs proliferate more rapidly in pHPL than in FBS, with no significant change in immunophenotype and differentiation capacity. However, not much is known about how pHPL affects the transcriptome of these cells.
This study investigated the effect of pHPL and FBS on the ASC transcriptome during in vitro serial expansion from passage 0 to passage 5 (P0 to P5). RNA was isolated from ASCs at each passage and hybridized to Affymetrix HuGene 2.0 ST arrays for gene expression analysis.
We observed that the transcriptome of ASCs expanded in pHPL (pHPL-ASCs) and FBS (FBS-ASCs) had the greatest change in gene expression at P2. Gene ontology revealed that genes upregulated in pHPL-ASCs were enriched for cell cycle, migration, motility, and cell-cell interaction processes, while those in FBS-ASCs were enriched for immune response processes. ASC transcriptomes were most homogenous from P2 to P5 in FBS and from P3 to P5 in pHPL. FBS- and pHPL-gene-specific signatures were observed, which could be used as markers to identify cells previously grown in either FBS or pHPL for downstream clinical/research applications. The number of genes constituting the FBS-specific effect was 3 times greater than for pHPL, suggesting that pHPL may be a milder supplement for cell expansion. A set of genes were expressed in ASCs at all passages and in both media. This suggests that a unique ASC in vitro transcriptomic profile exists that is independent of the passage number or medium used.
GO classification revealed that pHPL-ASCs are more involved in cell cycle processes and cellular proliferation when compared to FBS-ASCs, which are involved in more specialized or differentiation processes like cardiovascular and vascular development. This makes pHPL a potential superior supplement for expanding ASCs as they retain their proliferative capacity, remain untransformed and pHPL does not affect the genes involved in differentiation in specific developmental processes.
为了使脂肪来源的基质细胞（ASCs）在临床环境中安全使用，需要按照良好生产规范（GMP）进行制备。在一些人体临床试验中，用于体外扩增 ASCs 的胎牛血清（FBS）存在异种免疫和人畜共患病传播的风险。为了确保维持 GMP 标准，已将混合人血小板裂解物（pHPL）用作 FBS 的替代品。与 FBS 相比，pHPL 可使 ASCs 更快地增殖，而免疫表型和分化能力无明显变化。但是，对于 pHPL 如何影响这些细胞的转录组知之甚少。
本研究调查了 pHPL 和 FBS 在从第 0 代（P0）到第 5 代（P5）的体外连续传代过程中对 ASC 转录组的影响。在每个传代时从 ASCs 中分离 RNA，并将其杂交到 Affymetrix HuGene 2.0 ST 阵列上进行基因表达分析。
我们观察到，在 pHPL 中扩增的 ASCs（pHPL-ASCs）和 FBS 中扩增的 ASCs（FBS-ASCs）的转录组在 P2 时表达变化最大。GO 揭示了 pHPL-ASCs 中上调的基因富集于细胞周期、迁移、运动和细胞-细胞相互作用过程，而 FBS-ASCs 中上调的基因富集于免疫反应过程。在 FBS 中，从 P2 到 P5，ASC 转录组最均匀，而在 pHPL 中，从 P3 到 P5，ASC 转录组最均匀。观察到 FBS 和 pHPL 特异性的基因特征，可用于鉴定先前在 FBS 或 pHPL 中生长的细胞，用于下游临床/研究应用。构成 FBS 特异性效应的基因数量是 pHPL 的 3 倍，这表明 pHPL 可能是一种更温和的细胞扩增补充剂。一组基因在所有传代和两种培养基中均在 ASCs 中表达。这表明存在独立于使用的传代数或培养基的独特 ASC 体外转录组谱。
GO 分类显示，与参与心血管和血管发育等特定发育过程中的分化的 FBS-ASCs 相比，pHPL-ASCs 更参与细胞周期过程和细胞增殖。这使得 pHPL 成为扩增 ASCs 的潜在更好的补充剂，因为它们保留了增殖能力，保持未转化状态，并且 pHPL 不会影响特定发育过程中分化所涉及的基因。
