Generation of Spinal Motoneurons from Mouse Induced Pluripotent Stem Cells
Generation of Spinal Motoneurons from Mouse Induced Pluripotent Stem Cells
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发表时间：
2009
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通讯作者：
Atsushi Fujii;S. Chiba;E. Takada;Yuji Ueda;J. Shimizu;M. Beppu;N. Suzuki
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作者：
Atsushi Fujii;S. Chiba;E. Takada;Yuji Ueda;J. Shimizu;M. Beppu;N. Suzuki
严重的脊髓损伤和涉及运动神经元损失的疾病在临床环境中导致悲惨的结果，并且没有治疗可以逆转患者（疾病的最后阶段的残疾）。然而，细胞替代疗法是治疗脊髓运动神经元不可逆损伤和丧失的一种可能方法。胚胎干细胞已被考虑用作移植细胞和分析有效的细胞移植。最近，诱导多能干细胞iPS细胞已被认为是一种可行的替代来源，因为它们提供了容易获得的移植物，而不会造成免疫排斥的风险。此外，使用这些细胞不涉及伦理问题。在本研究中，我们建立了从小鼠iPS细胞产生脊髓运动神经元的方法，并在体外评估其神经功能。定量PCR分析显示，与未处理相比，音刺猬Shh加视黄酸处理使HB 9 mRNA表达水平加倍。Shh处理可明显诱导Tuj和HB 9双阳性脊髓运动神经元，Shh缺失可显著减少HB 9阳性神经元。为了增加脊髓运动神经元的诱导，我们还分析了骨形态蛋白BMP作为与Shh信号传导不同的诱导途径的效应。我们发现BMP-4抑制HB 9阳性神经元的诱导。因此，涉及通过使用重组受体蛋白阻断BMP的额外治疗导致HB 9阳性神经元的数量增加三倍。在KCL刺激下，产生的神经元表现出神经元特异性的Ca 2内流，与体外电活性神经元相似。因此，我们成功地从iPS细胞中产生了HB 9阳性的脊髓运动神经元，这可能是细胞替代的潜在来源。
Severe spinal cord injury and diseases involving motoneuron loss cause miserable outcomes in clinical settings, and no therapy can reverse the patients( disabilities in the final stages of the disease. Cell replacement therapy, however, is a possible approach for treating irreversible damage and loss of spinal motoneurons. Embryonic stem cells have been considered for use as graft cells and analyzed for e$cient cell transplantation. Recently, induced pluripotent stem iPS cells have been considered a viable alternative source because they provide easily accessible grafts without posing a risk of immunological rejection. Further more, the use of these cells does not involve ethical problems. In this study, we established methods to generate spinal motoneurons from mouse iPS cells and assessed their neural function in vitro. Quantita- tive PCR analysis revealed that sonic hedgehog Shh plus retinoic acid treatments doubled the level of HB9 mRNA expression as compared to no treatment. The treatment e$ciently induced Tuj- and HB9- double-positive spinal motoneurons, and deletion of Shh dramatically reduced HB9-positive neurons. To increase spinal motoneuron induction, we also analyzed the e#ects of bone morphologic protein BMP as an induction pathway di#erent from Shh signaling. We found that BMP-4 inhibited HB9-positive neuron induction. Therefore, additional treatment involving the blockade of BMP by using a recombinant receptor protein resulted in three times higher number of HB9-positive neurons. The generated neurons showed a neuron-specific Ca 2 influx upon KCL stimulation, similar to electrically active neurons in vitro. Thus, we successfully generated HB9-positive spinal motoneurons from iPS cells, which could be a potential source for cell replacement.