Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors

Hua Qin; An-Dong Zhao; Meng-Li Sun; Kui Ma; Xiao-Bing Fu

doi:10.1186/s40779-020-00284-2

Your Location：

Home >

Browse articles >

Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors

RESEARCH | Updated：2023-02-28

- Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors
- Military Medical Research Vol. 8, Issue 2, Pages: 162-173(2021)
- Affiliations：
  
  1.Research Center for Tissue Repair and Regeneration affiliated to the Medical Innovation Research Division and 4th Medical Center, PLA General Hospital and PLA Medical College, 28 Fu Xing Road, Haidian District, Beijing 100853, China
  2.Tianjin Medical University, Tianjin 300070, China
  3.PLA Key Laboratory of Tissue Repair and Regenerative Medicine and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, Beijing 100048, China
  4.Research Unit of Trauma Care, Tissue Repair and Regeneration, Chinese Academy of Medical Sciences, 2019RU051, Beijing 100048, China
- Author bio：
  
  * fuxiaobing@vip.sina.com
- Funds：
- DOI：10.1186/s40779-020-00284-2
  CLC：
- Published：2021-06
- Accepted：
Scan QR Code
Qin et al.: Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors. Mil Med Res, 2020, 7: 52.
DOI：

Qin et al.: Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors. Mil Med Res, 2020, 7: 52. DOI： 10.1186/s40779-020-00284-2.

摘要

Abstract

Background:

Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson’s disease. Several studies have reported the generation of dopaminergic (DA) neurons from mouse and human fibroblasts by ectopic expression of transcription factors

in which genetic manipulation is associated with potential risks.

Methods:

The small molecules and protein factors were selected based on their function to directly induce human fetal lung IMR-90 fibroblasts into DA neuron-like cells. Microscopical

immunocytochemical

and RT-qPCR analyses were used to characterize the morphology

phenotype

and gene expression features of the induced cells. The whole-cell patch-clamp recordings were exploited to measure the electrophysiological properties.

Results:

Human IMR-90 fibroblasts were rapidly converted into DA neuron-like cells after the chemical induction using small molecules and protein factors

with a yield of approximately 95% positive TUJ1-positive cells. The induced DA neuron-like cells were immunopositive for pan-neuronal markers MAP2

NEUN

and Synapsin 1 and DA markers TH

DDC

DAT

and NURR1. The chemical induction process did not involve a neural progenitor/stem cell intermediate stage. The induced neurons could fire single action potentials

which reflected partially the electrophysiological properties of neurons.

Conclusions:

We developed a chemical cocktail of small molecules and protein factors to convert human fibroblasts into DA neuron-like cells without passing through a neural progenitor/stem cell intermediate stage. The induced DA neuron-like cells from human fibroblasts might provide a cellular source for cell-based therapy of Parkinson’s disease in the future.

关键词

Keywords

references

Zhao A , Qin H , Fu X . What determines the regenerative capacity in animals? BioScience . 2016 ; 66 : 735 - 46 .

Riemens RJM , van den Hove DLA , Esteller M , Delgado-Morales R . Directing neuronal cell fate in vitro : achievements and challenges . Prog Neurobiol . 2018 ; 168 : 42 - 68 .

Yang N , Ng YH , Pang ZP , Südhof TC , Wernig M . Induced neuronal cells: how to make and define a neuron . Cell Stem Cell . 2011 ; 9 : 517 - 25 .

Stadhouders R , Filion GJ , Graf T . Transcription factors and 3D genome conformation in cell-fate decisions . Nature . 2019 ; 569 : 345 - 54 .

Xu J , Du Y , Deng H . Direct lineage reprogramming: strategies, mechanisms, and applications . Cell Stem Cell . 2015 ; 16 : 119 - 34 .

Qin H , Zhao A , Zhang C , Fu X . Epigenetic control of reprogramming and transdifferentiation by histone modifications . Stem Cell Rev . 2016 ; 12 : 708 - 20 .

Qin H , Zhao A , Fu X . Small molecules for reprogramming and transdifferentiation . Cell Mol Life Sci . 2017 ; 74 : 3553 - 75 .

Qin H , Zhao A , Ma K , Fu X . Chemical conversion of human and mouse fibroblasts into motor neurons . Sci China Life Sci . 2018 ; 61 : 1151 - 67 .

Qin H , Zhao A , Fu X . Chemical modulation of cell fates: in situ regeneration . Sci China Life Sci . 2018 ; 61 : 1137 - 50 .

Oh SM , Chang MY , Song JJ , Rhee YH , Joe EH , Lee HS , et al . Combined Nurr1 and Foxa2 roles in the therapy of Parkinson’s disease . EMBO Mol Med . 2015 ; 7 : 510 - 25 .

Della Valle F , Thimma MP , Caiazzo M , Pulcrano S , Celii M , Adroub SA , et al . Transdifferentiation of mouse embryonic fibroblasts into dopaminergic neurons reactivates LINE-1 repetitive elements . Stem Cell Rep . 2020 ; 14 : 60 - 74 .

Sheng C , Zheng Q , Wu J , Xu Z , Sang L , Wang L , et al . Generation of dopaminergic neurons directly from mouse fibroblasts and fibroblast-derived neural progenitors . Cell Res . 2012 ; 22 : 769 - 72 .

Kim J , Su SC , Wang H , Cheng AW , Cassady JP , Lodato MA , et al . Functional integration of dopaminergic neurons directly converted from mouse fibroblasts . Cell Stem Cell . 2011 ; 9 : 413 - 9 .

Pfisterer U , Kirkeby A , Torper O , Wood J , Nelander J , Dufour A , et al . Direct conversion of human fibroblasts to dopaminergic neurons . Proc Natl Acad Sci U S A . 2011 ; 108 : 10343 - 8 .

Caiazzo M , Dell’Anno MT , Dvoretskova E , Lazarevic D , Taverna S , Leo D , et al . Direct generation of functional dopaminergic neurons from mouse and human fibroblasts . Nature . 2011 ; 476 : 224 - 7 .

Liu X , Li F , Stubblefield EA , Blanchard B , Richards TL , Larson GA , et al . Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells . Cell Res . 2012 ; 22 : 321 - 32 .

Li X , Zuo X , Jing J , Ma Y , Wang J , Liu D , et al . Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons . Cell Stem Cell . 2015 ; 17 : 195 - 203 .

Hu W , Qiu B , Guan W , Wang Q , Wang M , Li W , et al . Direct conversion of normal and Alzheimer’s disease human fibroblasts into neuronal cells by small molecules . Cell Stem Cell . 2015 ; 17 : 204 - 12 .

Lim MS , Chang MY , Kim SM , Yi SH , Suh-Kim H , Jung SJ , et al . Generation of dopamine neurons from rodent fibroblasts through the expandable neural precursor cell stage . J Biol Chem . 2015 ; 290 : 17401 - 14 .

Colasante G , Lignani G , Rubio A , Medrihan L , Yekhlef L , Sessa A , et al . Rapid conversion of fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming . Cell Stem Cell . 2015 ; 17 ( 6 ): 719 - 34 .

Blanchard JW , Eade KT , Szucs A , Lo Sardo V , Tsunemoto RK , Williams D , et al . Selective conversion of fibroblasts into peripheral sensory neurons . Nat Neurosci . 2015 ; 18 : 25 - 35 .

He M , Zhang H , Li Y , Tian C , Tang B , Huang Y , Zheng J . Direct and selective lineage conversion of human fibroblasts to dopaminergic precursors . Neurosci Lett . 2019 ; 699 : 16 - 23 .

Cheng L , Hu W , Qiu B , Zhao J , Yu Y , Guan W , et al . Generation of neural progenitor cells by chemical cocktails and hypoxia . Cell Res . 2014 ; 24 : 665 - 79 .

Ladewig J , Mertens J , Kesavan J , Doerr J , Poppe D , Glaue F , et al . Small molecules enable highly efficient neuronal conversion of human fibroblasts . Nat Methods . 2012 ; 9 : 575 - 8 .

Arenas E , Denham M , Villaescusa JC . How to make a midbrain dopaminergic neuron . Development . 2015 ; 142 : 1918 - 36 .

Roessler R , Smallwood SA , Veenvliet JV , Pechlivanoglou P , Peng SP , Chakrabarty K , et al . Detailed analysis of the genetic and epigenetic signatures of iPSC-derived mesodiencephalic dopaminergic neurons . Stem Cell Rep . 2014 ; 2 : 520 - 33 .

Kriks S , Shim JW , Piao J , Ganat YM , Wakeman DR , Xie Z , et al . Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease . Nature . 2011 ; 480 : 547 - 51 .

Jiang H , Ren Y , Yuen EY , Zhong P , Ghaedi M , Hu Z , et al . Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells . Nat Commun . 2012 ; 3 : 668 .

Cooper O , Hargus G , Deleidi M , Blak A , Osborn T , Marlow E , et al . Differentiation of human ES and Parkinson’s disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid . Mol Cell Neurosci . 2010 ; 45 : 258 - 66 .

Andersson ER , Saltó C , Villaescusa JC , Cajanek L , Yang S , Bryjova L , et al . Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells . Proc Natl Acad Sci U S A . 2013 ; 110 : E602 - 10 .

Ratzka A , Baron O , Stachowiak MK , Grothe C . Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo . J Neurochem . 2012 ; 122 : 94 - 105 .

Castelo-Branco G , Wagner J , Rodriguez FJ , Kele J , Sousa K , Rawal N , et al . Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a . Proc Natl Acad Sci U S A . 2003 ; 100 : 12747 - 52 .

Chung S , Leung A , Han B-S , Chang M-Y , Moon J-I , Kim C-H , et al . Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway . Cell Stem Cell . 2009 ; 5 : 646 - 58 .

Decressac M , Volakakis N , Björklund A , Perlmann T . NURR1 in Parkinson disease—from pathogenesis to therapeutic potential . Nat Rev Neurol . 2013 ; 9 : 629 - 36 .

Kadkhodaei B , Alvarsson A , Schintu N , Ramsköld D , Volakakis N , Joodmardi E , et al . Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons . Proc Natl Acad Sci U S A . 2013 ; 110 : 2360 - 5 .

Lee HS , Bae EJ , Yi SH , Shim JW , Jo AY , Kang JS , et al . Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation, function, and cell survival . Stem Cells . 2010 ; 28 : 501 - 12 .

Kadkhodaei B , Ito T , Joodmardi E , Mattsson B , Rouillard C , Carta M , et al . Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons . J Neurosci . 2009 ; 29 : 15923 - 32 .

Wei P , Zhong C , Yang X , Shu F , Xiao S , Gong T , et al . Exosomes derived from human amniotic epithelial cells accelerate diabetic wound healing via PI3K-AKT-mTOR-mediated promotion in angiogenesis and fibroblast function . Burns Trauma . 2020 ; 8 : tkaa020 . https://doi.org/10.1093/burnst/tkaa020 https://doi.org/10.1093/burnst/tkaa020 .

Jiang H , Xu Z , Zhong P , Ren Y , Liang G , Schilling HA , et al . Cell cycle and p53 gate the direct conversion of human fibroblasts to dopaminergic neurons . Nat Commun . 2015 ; 6 ( 1 ): 1 - 14 .

Views

Downloads

CSCD

Alert me when the article has been cited

Submit

Tools

Publicity Resources

Efficient and rapid conversion of human astrocytes and ALS mouse model spinal cord astrocytes into motor neuron-like cells by defined small molecules

Gut microbiota helps identify clinical subtypes of Parkinson’s disease

Research on Synthetical 3 Factors Checking Method for the Ultimate Load Carrying Capacity of Steel Arch Bridges

Simulation Analysis on Static Performance of EP Solenoid Valves Based on ANSYS

Communication Technology in Two-out-of-Two Computer Interlocking System

Related Author

Hua Qin

Xiao-Bing Fu

Jing-Yi Wang

Rui Xie

Yun Feng

Min-Na Zhang

Le He

Bo Yang

Related Institution

Research Unit of Trauma Care, Tissue Repair and Regeneration, Chinese Academy of Medical Sciences

Department of Gastroenterology, the Affiliated Huai’an No.1 People’s Hospital of Nanjing Medical University

Department of Radiology, the Affiliated Huai’an No.1 People’s Hospital of Nanjing Medical University

Department of Laboratory Diagnosis

Changhai Hospital

⁰