Lim Ji-Hey, Koh Sehwon, Thomas Rachael, Breen Matthew, Olby Natasha J
Am J Vet Res. 2017 Mar;78(3):371-380. doi: 10.2460/ajvr.78.3.371.
OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs. ANIMALS 14 client-owned paraplegic dogs. PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively. RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.
目的 评估慢性截瘫犬组织来源的脂肪组织间充质干细胞（ADSCs）及ADSC来源的神经球样细胞簇（ADSC-NSCs）中的基因表达和DNA拷贝数。
动物 14只客户拥有的截瘫犬。
方法 在全身麻醉下采集背部皮下脂肪组织（<1 cm）；分离并培养ADSCs。将第三代ADSCs在神经细胞诱导培养基中培养以生成ADSC-NSCs。通过定量实时PCR检测法评估3只犬的ADSCs和ADSC-NSCs中间充质细胞表面标志物CD90和神经祖细胞标志物巢蛋白的相对基因表达；通过逆转录PCR检测法评估同一只犬的这些基因及各种神经谱系基因的表达。通过流式细胞术测定所有犬表达CD90、巢蛋白、胶质纤维酸性蛋白（GFAP）和微管蛋白β3 III类（TUJ1）蛋白的细胞百分比。分别采用阵列比较基因组杂交分析和免疫细胞化学评估法评估6只犬样本中的DNA拷贝数稳定性和14只犬的神经细胞分化情况。
结果 ADSCs和ADSC-NSCs表达神经细胞祖细胞和分化标志物；ADSC-NSCs表达GFAP和微管相关蛋白2，而ADSCs不表达。ADSC-NSCs中CD90和巢蛋白的相对基因表达在主观上高于ADSCs。表达巢蛋白、GFAP和TUJ1蛋白的ADSC-NSCs百分比显著高于ADSCs。从ADSC-NSCs中产生了表达神经元和神经胶质标志物的细胞，且所用方法未检测到DNA拷贝数不稳定情况。
结论及临床意义 结果表明ADSCs可能是犬神经祖细胞安全且与临床相关的自体来源。需要进一步研究以验证这些发现。
