Differentiation Potential of Human Induced Pluripotent Stem Cells (iPSCs) to Nucleus Pulposus-Like Cells in Vitro
Differentiation Potential of Human Induced Pluripotent Stem Cells (iPSCs) to Nucleus Pulposus-Like Cells in Vitro
复制标题
人诱导多能干细胞 (iPSC) 体外分化为髓核样细胞的潜力
DOI：
10.1055/s-0032-1319887
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发表时间：
2012
影响因子：
2.4
通讯作者：
J. Chen
中科院分区：
文献类型：
--
作者：
L. Jing;N. Christoforou;K. Leong;L. Setton;J. Chen
前言：将自体或异体细胞输送到突出或退变的椎间盘可促进组织再生和阻止退变1-4。从患者的体细胞来源的诱导多能干细胞(IPSCs)是一种有吸引力的细胞来源，具有分化为各种细胞类型的潜力。到目前为止，还没有研究表明人类IPSCs可以分化为髓核(NP)细胞。我们先前已经证明，人脐带间充质基质细胞和小鼠IPSCs在富含层粘连蛋白的3D培养系统中诱导分化后，都能够显示许多NP样细胞标志物6，7。本研究的目的是评估在这种类似的富层粘连蛋白环境中培养的人IPSCs是否也能表达独特的NP样细胞表型。材料与方法人胚胎真皮成纤维细胞经慢病毒载体(含反向四环素反式激活剂(M2rtTA)和强力霉素的多顺反子载体)瞬时诱导过表达转录因子(OCT4、SOX2、KLF4和MYC)，获得IPSCs。用多能标记试剂盒(OCT4、SOX2、SSEA-4、TRA1-60、TRA1-81和碱性磷酸酶、应用干细胞)进行免疫细胞化学。选定的IPSC菌落在PMEF饲养层上保持培养。细胞分化未分化的IPSCs(10 6个/孔，Transwell)接种于涂有Matrigel的孔中，形成富含层粘连蛋白的软基质6，7。细胞在从猪NP组织( + 1%ITS)8收集的条件培养液中培养28d。流式细胞术分析未分化的IPSCs与CD24、CD31、CD90、整合素亚单位a3(CD49c)、a6(CD49f)、b1(CD29)抗体共同孵育。对于转录因子(brachyury，OCT4)的细胞内表达，细胞在与一抗孵育之前被通透性。对细胞进行荧光分析(Accuri C6)，以量化阳性标记细胞的百分比。取免疫组织化学分化的细胞进行冰冻切片。组织学染色(HE染色和藏红花O染色)检测细胞形态和蛋白多糖的合成。免疫组织化学染色检测基质蛋白(II型胶原、LM511)、层粘连蛋白相关受体(CD239、整合素亚单位a3、a6、b1、b4)、其他标记物(vim、细胞角蛋白8(KRT8)、N-钙粘蛋白、CD24)和非NP标记物(I型胶原、E-钙粘蛋白)的表达。结果IPSCs在PMEF饲养层上增殖并形成集落，与胚胎干细胞相似(图1A)。在增殖过程中，IPSC克隆表达许多典型的人类多能标志物(如图1a中的SOX2、TRA1-60)，并表达整合素(α3、α6和β1亚单位)蛋白、其他NP标志物(CD2 4和短臂)以及MSCs标志物(CD2 9、CD90)。一旦在富含层粘连蛋白的软基质条件下培养，IPSCs就呈现出细胞聚集的形态。在分化第28天，IPSCs表达NP相关的基质蛋白(层粘连蛋白10，LM511；Saf O，图1B所示的II型胶原和蛋白多糖)，LM511特异性受体(CD239，整合素亚单位a3，a6和b4，图1B)，以及许多NP标志物(CD24，波形蛋白，细胞角蛋白8和N-钙粘蛋白，图1B)；值得注意的是，I型胶原和E-钙粘蛋白没有表达。图1(A)在饲养层上培养的未分化的IPSC菌落，并表达多能性标记(SOX2，TRA1-60，BAR = 100微米)。(B)在Matrigel培养系统(BAR = 50µm)中培养28天的分化IPSCs进行NP标志物、蛋白多糖(SAFO)、Col II、LM511、CD239、VIM、KRT8的免疫染色。结论本研究评价了一种新的细胞来源，可用于IVD的细胞治疗。结果表明，天然NP环境因子(富含层粘连蛋白的软基质和分泌的可溶性因子)能够促进人IPSC分化为具有许多NP样标记和形态的细胞类型。未来的研究将集中在从这些HiPSCs中选择NP基因的祖细胞用于IVD细胞治疗。由NIH R01AR057410、R01EB002263、R01AR047442、AOSpine基金会和FAMRI青年临床科学家奖支持的认可。我确认已经声明了本摘要中列出的所有作者的任何潜在利益冲突没有披露任何利益没有声明Nishimura等人。脊柱1998；23：1531 Okuma等人。《矫形外科研究杂志》2000；18：988 Meisel等人。欧洲脊柱杂志2006；15(S3)：397酒井。北美整形外科诊所。2011年；42：555山中。Cell 2009；137：13 Chon等人。Trans ORS 2011；36：600 Jing等人。Tans ORS 2012；37：31 PurMessur等人。关节炎研究疗法2011；13：R81
Introduction Autologous or allogeneic cell delivery to the herniated or degenerated intervertebral disk may promote tissue regeneration and arrest degeneration1–4. Induced pluripotent stem cells (iPSCs) derived from the patient's somatic cells represent an attractive cell source, having the potential to differentiate into various cell types5. To date, no studies have demonstrated that human iPSCs can differentiate into nucleus pulposus (NP) cells. We have previously shown that both human umbilical cord mesenchymal stromal cells and mouse iPSCs were able to display many NP-like cell markers after cultured in a laminin-rich 3D culture system for inducing differentiation6,7. The goal of this study is to evaluate whether human iPSCs cultured in this similar laminin-rich environment can also express a unique NP-like cell phenotype. Materials and Methods Cell Generation and Characterization iPSCs were generated from human embryonic dermal fibroblasts through transient inducible over-expression of transcription factors (OCT4, SOX2, KLF4 and MYC) by a lentiviral-based gene delivery system (a poly-cystronic vector with the Reverse Tetracycline Transactivator (M2rtTA) and doxycycline). For the derived colonies, immuno-cytochemistry was performed using a pluripotent markers kit (OCT4, SOX2, SSEA-4, TRA1-60, TRA1-81 and alkaline phosphatase, Applied StemCell). Selected iPSC colonies were maintained in culture upon a PMEF feeder layer. Cell Differentiation Undifferentiated iPSCs were seeded (106/well, Transwell) on wells precoated with Matrigel to generate a soft laminin-rich matrix6,7. Cells were cultured in conditioned medium collected from porcine NP tissues (DMEM + 1% ITS)8 for up to 28 days. Flow Cytometry Analysis Undifferentiated iPSCs were incubated with the antibodies against CD24, CD31, CD90, integrin subunits a3 (CD49c), a6 (CD49f), and b1 (CD29). For intracellular expression of transcription factors (Brachyury, OCT4), cells were permeabilized before incubation with primary antibodies. Cells were analyzed for fluorescence (Accuri C6) to quantify the percent of positively labeled cells. Immunohistochemistry Differentiated cells were harvested for cryo-sectioning. Cell morphology and proteoglycan synthesis was assessed by histological staining (H&E and Safranin O). Expression of NP markers was evaluated by immunostaining for matrix proteins (type II collagen (COL II), laminin 10 (LM511)), laminin-related receptors (CD239, integrin subunits a3, a6, b1, b4), and other markers (vimentin (VIM), cytokeratin 8 (KRT8), N-cadherin, CD24), as well as non-NP-markers (type I collagen, E-cadherin). Results iPSCs proliferated and formed colonies on PMEF feeder layers similar to embryonic stem cells (Fig. 1A). During proliferation, iPSC colonies expressed many typical human pluripotent markers (i.e. SOX2, TRA1-60 in Fig. 1A), and expressed integrin (α3, α6 and β1 subunit) proteins, other NP markers (CD24 and Brachyury), as well as MSC markers (CD29, CD90). Once cultured under soft laminin-rich matrix conditions, iPSCs were shown to adopt a cell clustering morphology. At day 28 of differentiation, iPSCs expressed NP-related matrix proteins (laminin 10, LM511; type II collagen and proteoglycans as indicated by Saf O, Fig. 1B), LM511 specific receptors (CD239, integrin subunit a3, a6 and b4, Fig. 1 B), as well as many NP markers (CD24, vimentin, cytokeratin 8 and N-cadherin, Fig. 1B); it is noteworthy that type I collagen and E-cadherin were not expressed. Figure 1 (A) Undifferentiated iPSC colonies cultured on a feeder layer and expressed pluripotency markers (SOX2, TRA1-60, bar = 100 µm). (B) Immunostaining for NP markers, proteoglycans (Saf O), Col II, LM511, CD239, VIM, KRT8 in differentiated iPSCs cultured for 28 days in a Matrigel system (bar = 50 µm). Conclusion Our study evaluated a novel cell source that has potential use for cellular therapy in the IVD. Results demonstrate that native NP environment factors (soft laminin-rich matrix and secreted soluble factors) are able to promote human iPSC differentiation into a cell type exhibiting many NP-like markers and morphology. Future studies will focus on selection of NP-genic progenitor cells from these hiPSCs for IVD cellular therapy. Acknowledgments Supported by NIH R01AR057410, R01EB002263, R01AR047442,AOSpine Foundation and FAMRI Young Clinical Scientist award. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Nishimura, et al. Spine 1998;23:1531 Okuma, et al. Journal of Orthopaedic Research 2000;18:988 Meisel, et al. European Spine Journal 2006;15(S3):397 Sakai. Orthopaedic Clinics of North America. 2011;42:555 Yamanaka. Cell 2009;137:13 Chon, et al. Trans ORS 2011;36:600 Jing, et al. Tans ORS 2012;37: 31 Purmessur, et al. Arthritis Research Therapy 2011;13:R81