Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
Department of Oncology, Georgetown University Medical School, Washington, DC, 20057, USA.
Stem Cell Res Ther. 2018 Jul 4;9(1):181. doi: 10.1186/s13287-018-0918-x.
Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium.
Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium.
Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers.
Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.
马的皮肤损伤经常导致缺乏角质细胞覆盖的慢性伤口，而角质细胞覆盖对于愈合至关重要。目前的方案中，马角质细胞的增殖有限，这限制了它们在再生医学中的应用。之前已经生产出了马诱导多能干细胞（eiPSC），并且可以将 eiPSC 分化为适合基于干细胞的皮肤构建的马角质细胞。然而，该过程技术上具有挑战性且耗时。本研究旨在评估条件重编程（CR）是否可以快速在未分化状态下扩增原代马角质细胞，但保留其正常分化和形成分层上皮的能力。
使用条件重编程分离并扩增了两个马角质细胞培养物。PCR 和 FISH 用于评估细胞的马起源，并进行核型分析以进行染色体计数。FACS 分析和免疫荧光用于确定马角质细胞的纯度及其增殖状态。使用三维气液界面法测试细胞分化和形成分层鳞状上皮的能力。
条件重编程是分离和扩增两个马角质细胞培养物的有效方法。细胞以 2.39 天/倍增的速度增殖，超过 40 个倍增。还开发了无饲养细胞的培养方法用于长期扩增。Rock 抑制剂对于饲养细胞和无饲养细胞条件以及维持增殖细胞的干细胞样状态都是至关重要的。PCR 和 FISH 验证了培养物中的马特异性标记。核型分析显示正常的马 64、XY 染色体。使用泛细胞角蛋白抗体的 FACS 显示出纯的角质细胞群体。当 ROCK 抑制剂被撤回并且细胞被转移到三维气液培养中时，它们形成了一个分化良好的分层鳞状上皮，其对终末分化标记呈阳性。
我们的结果证明，条件重编程是第一种允许快速和持续体外扩增原代马角质细胞的方法。这些无限供应的自体细胞可用于生成无免疫排斥风险的移植物。这为使用自体移植治疗难治性马伤口提供了机会。
