2012年12月07日 訊 /生物谷BIOON/ --誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cells, iPSCs)有潛力分化為體內(nèi)任何一種細(xì)胞,，從而提供一種用于藥物開(kāi)發(fā)和再生醫(yī)學(xué)的強(qiáng)大工具,。但是在實(shí)驗(yàn)室中大規(guī)模地誘導(dǎo)這些細(xì)胞可靠地選擇一種特定的命運(yùn)一直都是一個(gè)挑戰(zhàn)。如今,，來(lái)自新加坡A*STAR的一個(gè)干細(xì)胞研究人員小組開(kāi)發(fā)出一種細(xì)胞分化方法：在三維生物反應(yīng)器中增殖和擴(kuò)大iPSCs從而高效地產(chǎn)生神經(jīng)祖細(xì)胞(neural progenitor cell),。
論文通信作者Steve Oh和他的同事們利用他們之前為在三維懸浮系統(tǒng)中的小的固體顆粒表面上培養(yǎng)人胚胎干細(xì)胞開(kāi)發(fā)出的一種所謂的微載體平臺(tái)開(kāi)始研究。他們?yōu)槿薸PSCs優(yōu)化了這種技術(shù),，從而證實(shí)在每天更換兩次培養(yǎng)基的被稱作攪拌式反應(yīng)器的攪拌容器中,，包被蛋白的圓柱狀微載體能夠?qū)е轮鼐幊痰母杉?xì)胞增殖20倍。這種產(chǎn)量比任何其他報(bào)道的成批培養(yǎng)這些細(xì)胞的系統(tǒng)高得多,。
在正常條件下,，科學(xué)家們不得不在平直的培養(yǎng)皿中費(fèi)時(shí)費(fèi)力地操縱iPSCs以便形成更加特化的細(xì)胞。但是,，在這種新的三維平臺(tái)下,，只需簡(jiǎn)單地更換培養(yǎng)基，研究人員就可以高達(dá)85%的效率誘導(dǎo)iPSCs變成神經(jīng)祖細(xì)胞,。利用這種將細(xì)胞增殖和細(xì)胞分化整合在一起的過(guò)程,，每個(gè)接種的iPSC能夠產(chǎn)生333個(gè)神經(jīng)祖細(xì)胞,。作為比較，大多數(shù)科學(xué)家利用經(jīng)典的二維組織培養(yǎng)方法只能讓每個(gè)初始的干細(xì)胞產(chǎn)生53個(gè)神經(jīng)祖細(xì)胞,。
Oh說(shuō),，“這種二維方法費(fèi)時(shí)費(fèi)力，只能獲得10%的產(chǎn)量,，而且在不同實(shí)驗(yàn)室之間也是不同的,。基于微載體的培養(yǎng)方法為細(xì)胞生長(zhǎng)提供更加大的表面面積,，因而更加多的細(xì)胞能夠被加入到這種系統(tǒng)系統(tǒng)來(lái)增加集料尺寸和產(chǎn)量,。”
Oh和他的同事們也誘導(dǎo)這些神經(jīng)祖細(xì)胞進(jìn)一步分化為很多不同類型的腦細(xì)胞，包括三種主要的神經(jīng)細(xì)胞系：神經(jīng)元,、少突膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞,。Oh注意到，在未來(lái),，這樣的神經(jīng)元可能能夠被用來(lái)治療帕金森病之類的疾病,，而移植少突膠質(zhì)細(xì)胞可能能夠治療脊髓損傷。(生物谷Bioon.com)
doi: 10.1089/ten.tec.2012.0146
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Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells
Jo'an Bardy, Allen K. Chen, Yu Ming Lim, Selena Wu, Shunhui Wei, Han Weiping, Ken Chan, Shaul Reuveny, and Steve K.W. Oh
Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×106 cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1–60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×106 cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%–85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin+ neurons, GFAP+ astrocytes, and O4+ oligodendrocytes, showing that cells maintained their multilineage differentiation potential.
doi: 10.1016/j.scr.2011.04.007
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Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells
Allen Kuan-Liang Chena, , , Xiaoli Chena, , Andre Boon Hwa Chooa, , Shaul Reuvenyb, , Steve Kah Weng Oh
A variety of microcarriers may be used for the expansion of human embryonic stem cells (hESC) for cell therapy applications. This study investigated the effects of 10 types of microcarriers on hESC attachment efficiency, growth and pluripotency. High attachment efficiency was observed on uncoated microcarriers, however poor cell growth and/or gradual loss of pluripotency occurred during continuous passaging. Coating of the microcarriers with Matrigel resulted in higher cell yields and stable pluripotent states for at least three passages. Positively charged cylindrical cellulose microcarriers (DE52, DE53 and QA52) and large (190 μm) positively charged spherical microcarriers (Cytodex 1) exhibited high cell expansion potential and levels of pluripotency. Lower cell yields were obtained using smaller diameter spherical (65 μm and 10 μm) or macroporous beads. Instead of Matrigel, laminin coated microcarriers (DE53 and Cytodex 1) are capable of supporting the long term propagation and pluripotency of HES-2 and HES-3 cell lines. HES-2 cell line which was shown earlier to be shear resistant achieved similar cell growth and expression of pluripotent markers when cultured on both Matrigel (84% Tra-1-60, 1.43 × 106 cells/ml) and laminin (74% Tra-1-60, 1.37 × 106 cells/ml) coated microcarriers in spinner flasks. In contrast, HES-3 exhibited a decrease in cell yield, viability and pluripotent markers on laminin as compared with Matrigel coated microcarriers possibly due to shear sensitivity. Conventional microcarriers intended for propagation of mammalian cells are not suitable for long term propagation of hESC. Matrigel or laminin coating is essential for stable long term propagation of hESC on a variety of microcarriers
