Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model

Abstract

Autologous neural stem cells (NSCs) may offer a promising source for deriving dopaminergic (DA) cells for treatment of Parkinson's disease (PD).Methods: By using Sendai virus, human peripheral blood mononuclear cells (PBMNCs) were reprogrammed to induced NSCs (iNSCs), which were then differentiated to dopaminergic neurons in vitro. Whole-genome deep sequencing was performed to search for mutations that had accumulated during the reprogramming and expansion processes. To find the optimal differentiation stage of cells for transplantation, DA precursors obtained at various differentiation time points were tested by engraftment into brains of naïve immunodeficient mice. At last, the safety and efficacy of iNSC-derived DA precursors were tested by transplantation into the striatum of immunodeficient PD mouse models.Results: PBMNC-derived iNSCs showed similar characteristics to fetal NSCs, and were able to specifically differentiate to DA neurons with high efficiency in vitro. The sequencing data proved that no harmful SNVs, Indels and CNVs were generated during the reprogramming and expansion processes. DA precursors obtained between differentiation day 10 to 13 in vitro were most suitable for transplantation when a balanced graft survival and maturation were taken into account. Two weeks after transplantation of DA precursors into mouse PD models, the motor functions of PD mice started to improve, and continued to improve until the end of the experiments. No graft overgrowth or tumor was observed, and a significant number of A9-specific midbrain DA neurons were surviving in the striatum.Conclusion: This study confirmed the efficacy of iNSC-derived DA precursors in a mouse PD model, and emphasized the necessity of genomic sequencing and vigorous safety assessment before any clinical translation using iNSCs.

Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus

“…The use of an episomal vector is inexpensive and easily adaptable for reprogramming a variety of cell types, including iPSCs and iNSCs. Episomal vectors are eliminated from these iPSCs via several passages through cell divisions, which can take several months to achieve transgene-free iPSCs ( 30 , 32 ). However, Xu and colleagues indicated that piNPCs contained virtually undetectable amounts of EBNA-1, but episomal vector-transfected PFFs carried approximately 100 copies per cell, suggesting that EBNA-1 was not present in the piNPC genome ( 29 ).…”

“…These methods may result in insertional mutagenesis and the persistence or reactivation of transgenes. Moreover, therapeutic translation of this technique will require a thorough safety evaluation of mutations that occurred during the reprogramming process, as well as a fast derivation and differentiation strategy ( 32 ). A Sendai virus (SeV) vector can overcome these issues due to its single-stranded RNA virus propagating in the cytoplasm of infected cells that neither pass through a DNA phase nor integrate into the host genome, unlike other viruses.…”

Generation of Integration-Free Porcine Induced Neural Stem Cells Using Sendai virus

“…The use of episomal vector is very inexpensive and easily adaptable to reprogramming a variety of cell types, both iPSCs and iNSCs. Episomal vectors are eliminated from these iPSCs by several passages through cell divisions, which can take several months to achieve transgene-free iPSCs [30,42]. However, Xu and colleagues indicated that ipNPCs contained virtually undetectable amounts of EBNA-1, but episomal vector-transfected PFFs carried approximately 100 copies per cell, suggesting that EBNA-1 was not present in the ipNPC genome [27].…”

“…These methods may result in insertional mutagenesis and the persistence or reactivation of transgenes. Moreover, therapeutic translation of this technique will require a thorough safety evaluation of any mutations gained during the reprogramming process, as well as a fast derivation and differentiation strategy [30]. A Sendai virus (SeV) vector can overcome these issues owing to a single-stranded RNA virus propagating in the cytoplasm of infected cells that does neither pass a DNA phase nor integrate into the host genome, unlike the other viruses.…”

Impact of hydrogel stiffness on the induced neural stem cells modulation

“…iNSCs were generated from reprogrammed human blood mononuclear cells following our previous method and transfected EGFP. The iNSCs was identitied by NSC markers Sox1, Sox2, OLIG2, PAX6, NESTIN, GFAP and Ki67 (9). Subsequently, iNSCs were seeded in 0.6%, 0.8%, 1.0%, 1.2%, and 1.8% HA hydrogels in 48-well plates at the concentration of 2×10 5 cells per well.…”