Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy.
Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy.
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DOI：
10.1016/j.bbagen.2013.01.030
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发表时间：
2013-06
期刊：
影响因子：
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通讯作者：
Flavius C Pascut;S. Kalra;Vinoj T George;Nathan Welch;C. Denning;I. Notingher
中科院分区：
文献类型：
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作者：
Flavius C Pascut;S. Kalra;Vinoj T George;Nathan Welch;C. Denning;I. Notingher
背景干细胞体外分化的在线无标记监测仍然是干细胞研究中的主要挑战。在本文中，我们报告了使用拉曼显微光谱（RMS）来测量体外心原性分化过程中完整胚状体（EB）的时间和空间分辨分子变化。方法将人胚胎干细胞（hESC）聚集形成的EB在限定培养基中培养以诱导向心脏表型分化，并在拉曼显微镜下在专用微生物反应器中维持5天（分化第5天至第9天），每隔24小时记录一次空间分辨光谱。结果拉曼光谱显示，第7天EB自发搏动的开始与1340cm−1、1083cm−1、937cm−1、858cm−1、577cm−1和482cm−1处拉曼带强度的增加一致。这些条带对应的光谱图与通过相同EB的免疫荧光成像获得的心脏特异性α-辅肌动蛋白的表达具有高度正相关性。这里获得的光谱标记也与之前对单个活 hESC 衍生的 CM 进行的研究一致。结论这些拉曼谱带的强度分布可用于 EB 的无标记原位监测，以估计心源性分化的功效。一般意义由于时程拉曼光谱的获取不会影响 hESC 的活力或分化潜力，本研究证明了使用 RMS 进行在线非侵入性连续监测的可行性生物反应器培养系统内的过程。
BACKGROUNDOnline label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.METHODSEBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24h intervals.RESULTSThe Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340cm−1, 1083cm−1, 937cm−1, 858cm−1, 577cm−1and 482cm−1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.CONCLUSIONSThe intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.GENERAL SIGNIFICANCEAs the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.