兴奋性hiPSC源性类神经网络组织的构建

苏琪淞; 李戈; 许金海; 蒋斌; 位庆帅; 曾湘; 曾园山; 朱平

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001

您当前的位置：

首页 >

文章列表页 >

兴奋性hiPSC源性类神经网络组织的构建

基础研究 | 更新时间：2024-02-19

- 兴奋性hiPSC源性类神经网络组织的构建
- Construction of hiPSC-derived Excitatory Neural Network-like Tissue
- 中山大学学报（医学科学版） 2023年44卷第4期页码：625-633
- 作者机构：
  
  1.华南理工大学生物科学与工程学院，广东广州510006
  2.中山大学中山医学院组织学与胚胎学教研室，广东广州510080
  3.广东省心血管病研究所，南方医科大学附属广东省人民医院//广东省医学科学院，广东广州 510100
  4.医学研究部，南方医科大学附属广东省人民医院//广东省医学科学院，广东广州 510100
  5.广东省心脏病发病机制与精准防治重点实验室//广州市心脏病发病机制与防治重点实验室，广东广州 510100
  6.广东省脑功能与脑疾病重点实验室，广东广州 510080
- 作者简介：
  
  苏琪淞，研究方向：hiPSC源性类神经网络组织的构建，E-mail：QisongSu@126.com
  李戈，共同第一作者，研究方向：干细胞及组织工程技术修复中枢神经系统损伤，E-mail：lige@gdph.org.cn；
- 基金信息：
  
  国家自然科学基金(81891003;81974019;82271395;82001301);国家重点研发计划(2018YFA0108700);广东省特支计划杰出人才项目(2021JC06Y656);广东省科技计划项目(2020B1111170011;2022B1212010010);广东省自然科学基金(2023A1515030073);广东省粤澳青年双向交流项目(KD0120230024);广州市科技计划项目(202201000006);广东省人民医院登峰计划专项(DFJHBF2021111;KJ012020630;KY0120220133)
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001
  中图分类号： R318
- 纸质出版日期：2023-07-20，
  
  收稿日期：2023-02-08，
扫描看全文
苏琪淞,李戈,许金海等.兴奋性hiPSC源性类神经网络组织的构建[J].中山大学学报（医学科学版）,2023,44(04):625-633.

SU Qi-song,LI Ge,XU Jin-hai,et al.Construction of hiPSC-derived Excitatory Neural Network-like Tissue[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(04):625-633.
苏琪淞,李戈,许金海等.兴奋性hiPSC源性类神经网络组织的构建[J].中山大学学报（医学科学版）,2023,44(04):625-633. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001.

SU Qi-song,LI Ge,XU Jin-hai,et al.Construction of hiPSC-derived Excitatory Neural Network-like Tissue[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(04):625-633. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001.

摘要

目的

将人诱导多能干细胞源性神经前体细胞（hiPSC-NPCs）种植于去细胞视神经（ DON）上，在体外构建一种具有突触传递功能的类神经网络组织，为神经组织损伤的修复提供一种有效途径。

方法

通过定向诱导和组织工程技术，联合应用hiPSCs和3D DON支架，构建一种新的类神经网络组织。定向诱导人诱导多能干细胞（hiPSCs）向人神经前体细胞（ hNPCs）及神经元方向分化，使用免疫荧光染色鉴定细胞分化效率。制备3D DON支架，借助扫描电镜、Tunnel染色，鉴定支架形貌及细胞相容性。将诱导的hiPSC-NPCs种植于DON支架中，借助免疫荧光染色、扫描电镜、透射电镜和膜片钳对构建的类神经网络组织进行形态学观察和功能学鉴定。

结果

①免疫细胞化学染色结果提示，诱导的hiPSC-NPCs在体外大部分向神经元方向分化，成功构建了一种以神经元为主的神经网络。②扫描电镜和免疫组织化学结果提示，在体外成功构建了一种以兴奋性神经元为主的类神经网络组织。③免疫组织化学染色、透射电镜和膜片钳结果提示，构建的类神经网络组织能够传递兴奋性突触信息。

结论

利用天然来源的具有均匀孔道分布的DON生物支架材料和hiPSC-NPCs的有机结合，构建了一种以兴奋性神经元为主的具有突触传递功能的类神经网络组织，这种神经网络可作为脊髓损伤（spinal cord injury， SCI）等神经组织损伤后组织替换疗法的有利工具，具有广阔的临床应用前景。

Abstract

Objective

To construct a neural network-like tissue with the potential of synaptic formation in vitro by seeding human induced pluripotent stem cell-derived neural precursor cells （hiPSC-NPCs） on decellularized optic nerve （DON）， so as to provide a promising approach for repair of nerve tissue injury.

Methods

Through directional induction and tissue engineering technology， human induced pluripotent stem cells （hiPSCs） and 3D DON scaffolds were combined to construct neural network-like tissues. Then the hiPSCs were directionally induced into human neural precursor cells （hNPCs） and neurons. Immunofluorescence staining was used to identify cell differentiation efficiency. 3D DON scaffolds were prepared. Morphology and cytocompatibility of scaffolds were identified by scanning electron microscopy and Tunnel staining. Induced hiPSC-NPCs were seeded on DON scaffolds. Immunofluorescence staining， scanning electron microscopy， transmission electron microscopy and patch clamp were used to observe the morphology and functional identification of constructed neural network tissues.

Results

①The results of immunofluorescence staining suggested that most of hiPSC-NPCs differentiated into neurons in vitro. We had successfully constructed a neural network dominated by neurons. ② The results of scanning electron microscopy and immunohistochemistry suggested that a neural network-like tissue with predominating excitatory neurons in vitro was successfully constructed. ③The results of immunohistochemical staining， transmission electron microscopy and patch clamp indicated that the neural network-like tissue had synaptic transmission function.

Conclusion

A neural network-like tissue mainly composed of excitatory neurons has been constructed by the combination of natural uniform-channel DON scaffold and hiPSC-NPCs， which has the function of synaptic transmission. This neural network plays a significant role in stem cell derived replacement therapy， and offers a promising prospect for repair of spinal cord injury （SCI） and other neural tissue injuries.

关键词

人诱导多能干细胞源性神经前体细胞去细胞视神经突触兴奋性神经元类神经网络组织

Keywords

hiPSC-NPCsDONsynapseexcitatory neuronsneural network-like tissue

references

de Paulis S， Arlotta G， Calabrese M， et al. Postoperative intensive care management of aortic repair［J］. J Pers Med， 2022， 12（8）： 1351.

Pieczonka K， Fehlings MG. Incorporating combinatorial approaches to encourage targeted neural stem/progenitor cell integration following transplantation in spinal cord injury ［J］. Stem Cells Transl Med， 2023， 12（4）： 207-214.

Biswas A， Hutchins R. Embryonic stem cells［J］. Stem Cells Dev， 2007， 16（2）： 213-222.

Martin-Lopez M， Fernandez-Muñoz B， Canovas S. Pluripotent stem cells for spinal cord injury repair ［J］. Cells， 2021， 10（12）： 3334.

Takahashi K， Tanabe K， Ohnuki M， et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors［J］. Cell， 2007， 131（5）： 861-872.

Zheng Y， Gallegos CM， Xue H， et al. Transplantation of human induced pluripotent stem cell-derived neural progenitor cells promotes forelimb functional recovery after cervical spinal cord injury ［J］. Cells， 2022， 11（17）： 2765.

Khazaei M， Ahuja CS， Nakashima H， et al. Gdnf rescues the fate of neural progenitor grafts by attenuating notch signals in the injured spinal cord in rodents ［J］. Sci Transl Med， 2020， 12（525）： eaau3538.

Sun JH， Li G， Wu TT， et al. Decellularization optimizes the inhibitory microenvironment of the optic nerve to support neurite growth［J］. Biomaterials， 2020， 258： 120289.

Abad M， Mosteiro L， Pantoja C， et al. Reprogramming in vivo produces teratomas and ips cells with totipotency features［J］. Nature， 2013， 502（7471）： 340-345.

Suman S， Domingues A， Ratajczak J， et al. Potential clinical applications of stem cells in regenerative medicine［J］. Adv Exp Med Biol， 2019， 1201： 1-22.

Kriegstein A， Alvarez-Buylla A. The glial nature of embryonic and adult neural stem cells［J］. Annu Rev Neurosci， 2009， 32： 149-184.

Cao QL， Zhang YP， Howard RM， et al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage［J］. Exp Neurol， 2001， 167（1）： 48-58.

Barnabé-Heider F， Göritz C， Sabelström H， et al. Origin of new glial cells in intact and injured adult spinal cord［J］. Cell Stem Cell， 2010， 7（4）： 470-482.

Steward O， Sharp KG， Matsudaira Yee K. Long-distance migration and colonization of transplanted neural stem cells［J］. Cell， 2014， 156（3）： 385-387.

Castillo Bautista CM， Sterneckert J. Progress and challenges in directing the differentiation of human iPSCs into spinal motor neurons［J］. Front Cell Dev Biol， 2022， 10： 1089970.

Goparaju SK， Kohda K， Ibata K， et al. Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors［J］. Sci Rep， 2017， 7： 42367.

Andersen J， Revah O， Miura Y， et al. Generation of functional human 3d cortico-motor assembloids［J］. Cell， 2020， 183（7）： 1913-1929. e26.

Du ZW， Chen H， Liu H， et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells［J］. Nat Commun， 2015， 6： 6626.

Cutarelli A， Martínez-Rojas VA， Tata A， et al. A monolayer system for the efficient generation of motor neuron progenitors and functional motor neurons from human pluripotent stem cells［J］. Cells， 2021， 10（5）： 1127.

Chambers SM， Fasano CA， Papapetrou EP， et al. Highly efficient neural conversion of human es and ips cells by dual inhibition of smad signaling［J］. Nat Biotechnol， 2009， 27（3）： 275-280.

Niethamer TK， Larson AR， O'neill AK， et al. Ephrin-b1 mosaicism drives cell segregation in craniofrontonasal syndrome hiPSC-derived neuroepithelial cells［J］. Stem Cell Rep， 2017， 8（3）： 529-537.

Baarine M， Khan M， Singh A， et al. Functional characterization of iPSC-derived brain cells as a model for x-linked adrenoleukodystrophy［J］. PLoS One， 2015， 10（11）： e0143238.

Xu X， Tay Y， Sim B， et al. Reversal of phenotypic abnormalities by crispr/cas9-mediated gene correction in huntington disease patient-derived induced pluripotent stem cells［J］. Stem Cell Rep， 2017， 8（3）： 619-633.

De Becker A， ⅣRiet. Homing and migration of mesenchymal stromal cells： How to improve the efficacy of cell therapy？［J］. World J Stem Cells， 2016， 8（3）： 73-87.

Chen X， Zhao Y， Li X， et al. Functional multichannel poly（propylene fumarate）-collagen scaffold with collagen-binding neurotrophic factor 3 promotes neural regeneration after transected spinal cord injury［J］. Adv Healthc Mater， 2018， 7（14）： e1800315.

Li G， Zhang B， Sun JH， et al. An nt-3-releasing bioscaffold supports the formation of trkc-modified neural stem cell-derived neural network tissue with efficacy in repairing spinal cord injury［J］. Bioact Mater， 2021， 6（11）： 3766-3781.

Lai BQ， Che MT， Feng B， et al. Tissue-engineered neural network graft relays excitatory signal in the completely transected canine spinal cord［J］. Adv Sci， 2019， 6（22）： 1901240.

Sköld C， Levi R， Seiger A. Spasticity after traumatic spinal cord injury： Nature， severity， and location［J］. Arch Phys Med Rehabil， 1999， 80（12）： 1548-1557.

Dragojlovic N， Romanoski NL， Verduzco-Gutierrez M， et al. Prevalence and treatment characteristics of spastic hypertonia on first-time admission to acute inpatient rehabilitation ［J］. American journal of physical medicine & rehabilitation， 2022， 101（4）： 348-352.

Gong C， Zheng X， Guo F， et al. Human spinal gaba neurons alleviate spasticity and improve locomotion in rats with spinal cord injury［J］. Cell Rep， 2021， 34（12）： 108889.

彭历芝，位庆帅，马瑗锾，等. 构建具有突触传递潜能的iPSC源性抑制性神经网络组织［J］. 中山大学学报（医学科学版）， 2023， 44（1）： 18-25.

Peng LZ， Wei QS， Ma YH， et al. Construction of iPSC-derived inhibitory neural network tissue with synaptic transmission potentials［J］. J Sun Yat-sen Univ（Med Sci）， 2023， 44（1）： 18-25.

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

暂无数据