Division of Endocrine Surgery, Department of Surgery, University of Michigan Health System, Ann Arbor, Michigan, USA.
Tissue Eng Part C Methods. 2011 Nov;17(11):1051-9. doi: 10.1089/ten.tec.2011.0170. Epub 2011 Jul 28.
Replacement of a diseased organ with an autologously derived tissue is an ideal therapy for some medical problems. However, it is difficult to recreate many adult human tissues in vitro due to the functionally necessary architecture of most organs and the lack of understanding of methods to direct the development of the organ of interest. The parathyroid gland is ideal for in vitro organ development because this gland is relatively simple, is transplantable, and is commonly affected by a surgical complication rather than an autoimmune disease. We have investigated thymus as a source of autologous endoderm and parathyroid-like precursor cells. Human thymus cells were treated with a differentiation protocol we developed with human embryonic stem cells (The Bingham Protocol) that utilizes timed exposures to Activin A and soluble Sonic hedgehog (Shh). We incrementally changed the protocol to optimize the differentiation of the thymus cells into parathyroid-like cells. The final protocol used 50 ng/mL Activin A and 100 ng/mL Shh over 13 weeks. The differentiated cells expressed the parathyroid markers parathyroid hormone (PTH), calcium sensing receptor, chemokine receptor type-4 (CXCR4), and chorian-specific transcription factor (GCM2) as measured by reverse transcription-polymerase chain reaction and PTH enzyme-linked immunosorbent assay. Cultured thymus cells without Activin A or Shh exposure did not secrete PTH nor express similar markers. The differentiated cells released PTH, which was suppressed in response to increased calcium concentration. The chemically differentiated cells did not form tumors in immune-compromised mice. Our protocol recreated cells with markers of parathyroid tissue that responded as parathyroid cells to physiologic stimuli. This approach is a further step toward a strategy to restore parathyroid function using autologous cells that were directed to differentiate by nongenetic in vitro manipulation.
用自体衍生组织替代病变器官是治疗某些医学问题的理想疗法。然而，由于大多数器官的功能必需结构以及缺乏指导感兴趣器官发育的方法的理解，因此很难在体外重建许多成人组织。甲状旁腺是体外器官发育的理想选择，因为这种腺体相对简单，可以移植，并且通常受到手术并发症的影响，而不是自身免疫性疾病的影响。我们研究了胸腺作为自体内胚层和甲状旁腺样前体细胞的来源。用人胚胎干细胞（宾厄姆方案）开发的分化方案处理人胸腺细胞，该方案利用激活素 A 和可溶性 Sonic hedgehog (Shh) 的定时暴露。我们逐步改变方案，以优化胸腺细胞向甲状旁腺样细胞的分化。最终方案使用 50ng/mL 的激活素 A 和 100ng/mL 的 Shh 持续 13 周。分化的细胞通过逆转录聚合酶链反应和甲状旁腺激素酶联免疫吸附测定表达甲状旁腺标记物甲状旁腺激素 (PTH)、钙敏感受体、趋化因子受体型-4 (CXCR4) 和 chorian 特异性转录因子 (GCM2)。未暴露于激活素 A 或 Shh 的培养胸腺细胞不分泌 PTH 也不表达类似标记物。分化的细胞释放 PTH，而增加的钙浓度会抑制其释放。化学分化的细胞在免疫缺陷小鼠中未形成肿瘤。我们的方案重现了具有甲状旁腺组织标记物的细胞，这些细胞对生理刺激的反应与甲状旁腺细胞相似。这种方法是使用通过非遗传体外操作指导分化的自体细胞恢复甲状旁腺功能的策略的进一步步骤。
