12月6日,，Cell Research在線發(fā)表了中科院上海生科院健康所楊黃恬研究組的研究成果，研究人員在誘導(dǎo)多能干細(xì)胞向心肌細(xì)胞分化研究方面取得新進(jìn)展,。
誘導(dǎo)多能干細(xì)胞（induced pluripotent stem cells, iPSCs）分化的心肌細(xì)胞對于藥物篩選、心肌再生醫(yī)學(xué)及其心臟發(fā)育生物學(xué)的研究均具有重要意義，并是研究人類心臟疾病提供了獨(dú)特的體外模型,。然而iPSCs的自發(fā)心肌細(xì)胞分化效率極低且分化的心肌細(xì)胞功能也相對的不成熟，各系之間的分化潛能差異大,，因此,，明確其定向分化的關(guān)鍵環(huán)節(jié)和調(diào)控機(jī)制，并建立穩(wěn)定高效、經(jīng)濟(jì)簡便地誘導(dǎo)iPSCs分化為所需的功能心肌細(xì)胞是研究其應(yīng)用價值的關(guān)鍵前提之一,。
健康科學(xué)研究所,、中國科學(xué)院干細(xì)胞生物學(xué)重點(diǎn)實(shí)驗室分子心臟學(xué)課題組的博士研究生曹楠等在楊黃恬研究員的指導(dǎo)下，采用中科院動物所,、上海生命科學(xué)院生化與細(xì)胞所,、健康所、廣州生物醫(yī)藥與健康院建立的11株不同的iPSCs系統(tǒng)篩選了16種心肌細(xì)胞分化誘導(dǎo)物,，發(fā)現(xiàn)只有抗壞血酸(ascorbic acid, AA) 表現(xiàn)出一致和高效的誘導(dǎo)iPSCs向心肌細(xì)胞分化作用,。通過比較不同濃度和不同分化時段AA的處理，建立了簡單經(jīng)濟(jì)的高效誘導(dǎo)iPSCs向心肌細(xì)胞分化的體系,，可將小鼠和人iPSCs向心肌細(xì)胞的分化效率分別提高了約7.3倍和30.2倍,，并明顯縮小了不同iPSCs系間，包括不具有自發(fā)心肌細(xì)胞分化潛能的iPSCs間心肌細(xì)胞的分化差異,。進(jìn)一步的機(jī)制研究發(fā)現(xiàn),，AA促進(jìn)心肌細(xì)胞分化作用是通過增強(qiáng)胞外膠原的分泌，從而激活MEK-ERK1/2 pathway,，進(jìn)而特異性地促進(jìn)心肌前體細(xì)胞增殖而實(shí)現(xiàn)的,。
此外，通過結(jié)構(gòu)和功能分析發(fā)現(xiàn),，AA誘導(dǎo)產(chǎn)生的心肌細(xì)胞表現(xiàn)出更為成熟的電活動和鈣活動特征,，其肌絲排列更為規(guī)律有序，對于重要的心肌細(xì)胞功能調(diào)控信號的反應(yīng)性也明顯增強(qiáng),，表明AA促進(jìn)了iPSCs分化的心肌細(xì)胞的成熟度,。該研究發(fā)現(xiàn)進(jìn)一步揭示了細(xì)胞外微環(huán)境對于多能干細(xì)胞向心肌細(xì)胞分化和心肌前體細(xì)胞的增殖具有重要的調(diào)控作用，所建立的簡便經(jīng)濟(jì)的高效誘導(dǎo)小鼠和人iPSCs向心肌細(xì)胞分化的體系,，有助于iPSCs在心臟疾病發(fā)生,、藥物篩選以及心臟再生醫(yī)學(xué)方面的應(yīng)用研究。（生物谷Bioon.com）
doi:10.1038/cr.2011.195
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Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells
Nan Cao, Zumei Liu, Zhongyan Chen, Jia Wang, Taotao Chen, Xiaoyang Zhao, Yu Ma, Lianju Qin, Jiuhong Kang, Bin Wei, Liu Wang, Ying Jin and Huang-Tian Yang
Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.
