Bingham Eve L, Cheng Shih-Ping, Woods Ignatoski Kathleen M, Doherty Gerard M
Department of Surgery, Endocrine Surgery Research Laboratory, University of Michigan, Ann Arbor, Michigan 48109, USA.
Stem Cells Dev. 2009 Sep;18(7):1071-80. doi: 10.1089/scd.2008.0337.
Iatrogenic hypoparathyroidism is the most common complication of cervical endocrine surgery. Current management is limited and palliative. As the molecular steps in parathyroid development have been defined, they may be replicable in vitro, with a goal of cellular replacement therapy. Human embryonic stem cell (hESC) lines were investigated as a model for parathyroid regeneration in vitro. BG01 was selected as a model based on expression of genes of interest in embryoid bodies (EBs). Established strategies for mouse embryonic stem cell differentiation into definitive endoderm were modified and extended to maximize the expression of definitive markers of parathyroid development. The optimal approach included the use of Activin A at 100 ng/mL with BG01 cells grown on murine embryonic fibroblasts for 5 days under conditions of increasing serum concentration. After 5 days, the cells were allowed to mature further in tissue culture without murine fibroblasts but with continuous Activin A. Our strategy produced differentiated cell cultures that expressed intermediate markers of endoderm and parathyroid development (CXCR4, EYA1, Six1, and Pax1), as well as markers of committed parathyroid precursors or developed parathyroid glands (glial cell missing-2 [Gcm2], CCL21, calcium sensing receptor [CaSR], and parathyroid hormone [PTH]). We further characterized the cells by testing conditioned medium from various time points in our differentiation scheme for the presence of PTH. We found that by keeping the cells in culture 2 weeks after the withdrawal of Activin A, the cells were able to produce PTH. Further in vivo work will be needed to demonstrate proper functionality of the cells developed in this way.
医源性甲状旁腺功能减退是颈部内分泌手术最常见的并发症。目前的治疗方法有限且只能缓解症状。由于甲状旁腺发育的分子步骤已明确，或许可在体外复制，目标是进行细胞替代疗法。研究了人胚胎干细胞（hESC）系作为甲状旁腺体外再生的模型。基于胚胎体（EBs）中感兴趣基因的表达，选择BG01作为模型。对已有的将小鼠胚胎干细胞分化为确定内胚层的策略进行了修改和扩展，以最大化甲状旁腺发育确定标志物的表达。最佳方法包括在血清浓度逐渐增加的条件下，将100 ng/mL的激活素A与在小鼠胚胎成纤维细胞上生长的BG01细胞一起培养5天。5天后，让细胞在没有小鼠成纤维细胞但有持续激活素A的组织培养中进一步成熟。我们的策略产生了表达内胚层和甲状旁腺发育中间标志物（CXCR4、EYA1、Six1和Pax1）以及已确定的甲状旁腺前体或发育中的甲状旁腺标志物（神经胶质细胞缺失-2 [Gcm2]、CCL21、钙敏感受体 [CaSR] 和甲状旁腺激素 [PTH]）的分化细胞培养物。我们通过检测分化方案中各个时间点的条件培养基中是否存在PTH，进一步对细胞进行了表征。我们发现，在去除激活素A后将细胞在培养中保留2周，这些细胞能够产生PTH。还需要进一步的体内研究来证明以这种方式培养的细胞的正常功能。
