Embryoid Body Formation from Mouse and Human Pluripotent Stem Cells for Transplantation to Study Brain Microenvironment and Cellular Differentiation

Guerra-Crespo, Magdalena; Collazo-Navarrete, Omar; Ramos-Acevedo, Rodrigo; Morato-Torres, Carmen Alejandra; Schüle, Birgitt

doi:10.1007/7651_2021_433

Cited by 3 publications

(2 citation statements)

References 24 publications

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“…This protocol has previously been documented for its capacity to yield highly efficient and reproducible floor-plate dopaminergic progenitors and facilitate dopaminergic differentiation [ 36 ]. Firstly, hiPSCs were thawed in 0.1% gelatin-coated 6-well culture plates (Sigma Aldrich, St. Louis, MO, USA, and Thermo Fisher Scientific, respectively) with mouse embryonic fibroblasts as feeder cells in hiPSC medium (DMEM-F12, KnockOut Serum Replacement, GlutaMAX, nonessential amino acids, penicillin–streptomycin, β-mercaptoethanol, and Fgf2) [ 72 ]. Once colonies were present, they were transitioned to a feeder-free culture.…”

Section: Methodsmentioning

confidence: 99%

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson’s Disease Animal Model

Bernal-Conde,

Peña-Martínez,

Morato-Torres

et al. 2024

Life

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Parkinson’s disease (PD) caused by SNCA gene triplication (3XSNCA) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3XSNCA and its absence is crucial. This study investigates the differentiation of human induced pluripotent stem cell (hiPSC)-derived floor-plate progenitors into dopaminergic neurons. Three different genotypes were evaluated in this study: patient-derived hiPSCs with 3XSNCA, a gene-edited isogenic line with a frame-shift mutation on all SNCA alleles (SNCA 4KO), and a normal wild-type control. Our aim was to assess how the substantia nigra pars compacta (SNpc) microenvironment, damaged by 6-hydroxydopamine (6-OHDA), influences tyrosine hydroxylase-positive (Th+) neuron differentiation in these genetic variations. This study confirms successful in vitro differentiation into neuronal lineage in all cell lines. However, the SNCA 4KO line showed unusual LIM homeobox transcription factor 1 alpha (Lmx1a) extranuclear distribution. Crucially, both 3XSNCA and SNCA 4KO lines had reduced Th+ neuron expression, despite initial successful neuronal differentiation after two months post-transplantation. This indicates that while the SNpc environment supports early neuronal survival, SNCA gene alterations—either amplification or knock-out—negatively impact Th+ dopaminergic neuron maturation. These findings highlight SNCA’s critical role in PD and underscore the value of hiPSC models in studying neurodegenerative diseases.

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Section: Methodsmentioning

confidence: 99%

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson’s Disease Animal Model

Bernal-Conde,

Peña-Martínez,

Morato-Torres

et al. 2024

Life

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“…The detached colonies were transferred to ultra-low adhesion culture wells (Sigma-Aldrich) and cultured for 4 days with E6 medium supplemented with 15% inactivated fetal bovine serum (Thermo Fisher). On the fourth day, EBs were rinsed, disaggregated, and resuspended in 5 µl of phosphate-buffered saline (PBS, Thermo Fisher) for transplantation [22].…”

Section: Embryoid Body Culturementioning

confidence: 99%

Human embryonic stem cells overexpressing dopaminergic transcription factors survive and differentiate in the substantia nigra in vivo

Ramos-Acevedo

Morato-Torres

Bernal-Conde

et al. 2022

Preprint

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Background: Degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson's disease (PD) is responsible for motor and cognitive impairment. Replacing the dopaminergic cell population in the SNpc to restore normal dopamine levels is a potential therapeutic approach. However, improving neuronal integration still requires a reliable cell source for transplantation and a profound understanding of the effects of the local microenvironment on transplanted cells. We have previously shown that embryoid bodies (EBs)-derived cells from mouse embryonic stem cells overexpressing the dopaminergic transcription factor Lmx1a engrafted into SNpc develop tyrosine hydroxylase (TH)-positive phenotype. In the present work, we transplanted EBs-derived cells from genetically engineered human embryonic stem cells (hESCs), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO). We determined their potential to differentiate into TH-expressing neurons in the SNpc of an in vivo PD model. Methods: EBs-derived cells from genetically the engineered hESCs-LFO cell line were transplanted, and their neuronal differentiation potential was determined in the SNpc of an in vivo PD model with 6-hydroxy dopamine (6-OHDA). Three rat groups were designed as follows: Untreated (healthy rats), sham (rats administered with saline solution), and 6-OHDA (rats lesioned with 6-OHDA). A one-way ANOVA test was performed for statistical analysis. Results: Neural rosettes, a fundamental developmental hallmark of neuroepithelial tissue, were found at 7 and 15 days post-transplantation (dpt) in ~ 70% of the transplanted brains in all three conditions: Untreated, sham, and 6-OHDA. The majority of the neural rosettes corresponded to the lumen formation stage. In comparison, no graft survival was observed in EB transplants derived from unmodified hESCs. Interestingly, at 30 dpt, hESC-LFO engrafted cells showed neuronal morphology and positive immunolabeling for TH in all the brains exhibiting surviving transplants: 10% 6-OHDA rats, 0% sham, and 100% untreated rats. Conclusions: Overall, our results show that overexpression of LFO factors favors short-term survival while strongly initiating neural differentiation of hESC-derived cells in SNpc surviving grafts by forming neural rosettes and differentiating into TH-positive cells.

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Embryoid Body Cells from Human Embryonic Stem Cells Overexpressing Dopaminergic Transcription Factors Survive and Initiate Neurogenesis via Neural Rosettes in the Substantia Nigra

et al. 2023

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Transplantation of immature dopaminergic neurons or neural precursors derived from embryonic stem cells (ESCs) into the substantia nigra pars compacta (SNpc) is a potential therapeutic approach for functional restitution of the nigrostriatal pathway in Parkinson’s disease (PD). However, further studies are needed to understand the effects of the local microenvironment on the transplanted cells to improve survival and specific differentiation in situ. We have previously reported that the adult SNpc sustains a neurogenic microenvironment. Non-neuralized embryoid body cells (EBCs) from mouse ESCs (mESCs) overexpressing the dopaminergic transcription factor Lmx1a gave rise to many tyrosine hydroxylase (Th+) cells in the intact and damaged adult SNpc, although only for a short-term period. Here, we extended our study by transplanting EBCs from genetically engineered naive human ESC (hESC), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO), in the SNpc. Unexpectedly, no graft survival was observed in wild-type hESC EBCs transplants, whereas hESC-LFO EBCs showed viability in the SNpc. Interestingly, neural rosettes, a developmental hallmark of neuroepithelial tissue, emerged at 7- and 15- days post-transplantation (dpt) from the hESC-LFO EBCs. Neural rosettes expressed specification dopaminergic markers (Lmx1a, Otx2), which gave rise to several Th+ cells at 30 dpt. Our results suggest that the SNpc enables the robust initiation of neural differentiation of transplanted human EBCs prompted to differentiate toward the midbrain dopaminergic phenotype.

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Embryoid Body Formation from Mouse and Human Pluripotent Stem Cells for Transplantation to Study Brain Microenvironment and Cellular Differentiation

Cited by 3 publications

References 24 publications

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson’s Disease Animal Model

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson’s Disease Animal Model

Human embryonic stem cells overexpressing dopaminergic transcription factors survive and differentiate in the substantia nigra in vivo

Embryoid Body Cells from Human Embryonic Stem Cells Overexpressing Dopaminergic Transcription Factors Survive and Initiate Neurogenesis via Neural Rosettes in the Substantia Nigra

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