Santamaria Pere, Rodriguez-Piza Ignacio, Clemente-Casares Xavier, Yamanouchi Jun, Mulero-Perez Lola, Aasen Trond, Raya Angel, Izpisua Belmonte Juan Carlos
Center of Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain.
Rev Diabet Stud. 2010 Summer;7(2):158-67. doi: 10.1900/RDS.2010.7.158. Epub 2010 Aug 10.
Human embryonic stem (hES) cells can be differentiated into pancreatic endoderm structures in vitro. The study was performed to determine whether induced pluripotent stem (iPS) cells can be differentiated into similar structures with comparable efficiency.
We compared the ability of hES cells and iPS cells derived from human epidermal keratinocytes to progressively differentiate into pancreatic endoderm. Human foreskin keratinocytes were reprogrammed to pluripotency by transduction with retroviruses encoding Oct4, Sox2, and Klf4. The resulting keratinocyte-derived iPS (KiPS) cell lines and a hES cell line were subjected to a modified pancreatic endoderm differentiation protocol. Cells and embryoid-body structures derived from both hES and KiPS cells were compared at different stages of development for expression of stem cell and differentiation markers, including Sox2, Oct4, Mixl1, Brachyury, Gsc, FoxA2, Sox17, Hnf4α, Hnf1β, Nkx2.2, Nkx6.1, Hex, Isl1, Pdx1, and Slc2A, via Taqman real-time PCR, flow-cytometry, and/or immunocytochemistry.
hES cells and KiPS cells expressed similar levels of the stem cell factors Sox2 and Oct4. Upon differentiation, both cell types underwent remarkably similar changes in gene expression. They acquired the definitive endoderm markers Sox17 and FoxA2. Most Sox17+ and FoxA2+ cells co-expressed Hnf4α and Hnf1β, found in the primitive gut tube, a pancreas precursor. Most FoxA2+ cells were also Pdx1+, and many expressed Nkx2.2, Nkx6.1, and Isl1.
Keratinocyte-derived iPS cells can be differentiated into pancreatic endoderm, and the efficiency of this process is comparable to that seen for hES cells. Thus keratinocytes have the potential to serve as a source of patient-specific pancreatic endoderm for transplantation.
人胚胎干细胞（hES）可在体外分化为胰腺内胚层结构。本研究旨在确定诱导多能干细胞（iPS）是否能以相当的效率分化为类似结构。
我们比较了hES细胞和源自人表皮角质形成细胞的iPS细胞逐步分化为胰腺内胚层的能力。通过用编码Oct4、Sox2和Klf4的逆转录病毒转导，将人包皮角质形成细胞重编程为多能性。将所得的角质形成细胞衍生的iPS（KiPS）细胞系和一个hES细胞系进行改良的胰腺内胚层分化方案。通过Taqman实时PCR、流式细胞术和/或免疫细胞化学，比较了hES和KiPS细胞来源的细胞及胚状体结构在不同发育阶段干细胞和分化标志物的表达，这些标志物包括Sox2、Oct4、Mixl1、Brachyury、Gsc、FoxA2、Sox17、Hnf4α、Hnf1β、Nkx2.2、Nkx6.1、Hex、Isl1、Pdx1和Slc2A。
hES细胞和KiPS细胞表达相似水平的干细胞因子Sox2和Oct4。分化后，两种细胞类型在基因表达上经历了非常相似的变化。它们获得了确定内胚层标志物Sox17和FoxA2。大多数Sox17+和FoxA2+细胞共表达Hnf4α和Hnf1β，这两种蛋白存在于原始肠管（胰腺前体）中。大多数FoxA2+细胞也是Pdx1+，许多细胞还表达Nkx2.2、Nkx6.1和Isl1。
角质形成细胞衍生的iPS细胞可分化为胰腺内胚层，且该过程的效率与hES细胞相当。因此，角质形成细胞有潜力作为移植用的患者特异性胰腺内胚层的来源。
