Robust and Reproducible Generation of Induced Neural Stem Cells from Human Somatic Cells by Defined Factors

Abstract

Background and Objectives: Recent studies have described direct reprogramming of mouse and human somatic cells into induced neural stem cells (iNSCs) using various combinations of transcription factors. Although iNSC technology holds a great potential for clinical applications, the low conversion efficiency and limited reproducibility of iNSC generation hinder its further translation into the clinic, strongly suggesting the necessity of highly reproducible method for human iNSCs (hiNSCs). Thus, in orderto develop a highly efficient and reproducible protocol for hiNSC generation, we revisited the reprogramming potentials of previously reported hiNSC reprogramming cocktails by comparing the reprogramming efficiency of distinct factor combinations including ours. Methods: We introduced distinct factor combinations, OSKM (OCT4+SOX2+KLF4+C-MYC), OCT4 alone, SOX2 alone, SOX2+HMGA2, BRN4+SKM+SV40LT (BSKM LT ), SK LT , SM LT , and SKM LT and performed comparative analysis of reprogramming potentials of distinct factor combinations in hiNSC generation. Results: Here we show that ectopic expression of five reprogramming factors, BSKM LT leads the robust hiNSC generation (>80 folds enhanced efficiency) from human somatic cells compared with previously described factor combinations. With our combination, we were able to observe hiNSC conversion within 7 days of transduction. Throughout further optimization steps, we found that both BRN4 and KLF4 are not essential for hiNSC conversion. Conclusions: Our factor combination could robustly and reproducibly generate hiNSCs from human somatic cells with distinct origins. Therefore, our novel reprogramming strategy might serve as a useful tool for hiNSC-based clinical application.

The occurrence and development of induced pluripotent stem cells

“…To date, iPSCs have been used to study various neurological diseases ( Kwak et al, 2020 ; Han et al, 2021 ), including amyotrophic lateral sclerosis (ALS) ( Dimos et al, 2008 ; Kabashi et al, 2010 ; Egawa et al, 2012 ), Alzheimer’s disease (AD) ( Táncos et al, 2016a ; Chandrasekaran et al, 2016 ; Ochalek et al, 2016 ; Hernández-Sapiéns et al, 2020 ; Raska et al, 2021a ; Raska et al, 2021b ), and Parkinson’s disease (PD) ( Ma et al, 2017a ; Wang et al, 2017a ; Ma et al, 2017b ; Ma et al, 2017c ; Ma et al, 2017d ). Dimos et al obtained the first human iPSCs from middle-aged and elderly ALS patients ( Dimos et al, 2008 ), and Egawa et al demonstrated that motor neurons generated from iPSCs from patients with TDP-43 mutations can form cytoplasmic aggregates typical of postdeath ALS neurons ( Egawa et al, 2012 ).…”

Research progress of the transcription factor Brn4 (Review)

“…2). These cells, after being transplanted into the stem cell pool in the adult mouse brain, exhibited true pluripotency in terms of proliferation and differentiation into neurons and glial cells (81,82). The reprogramming efficiency was further improved by the addition of E47 or transcription factor 3 (83).…”

Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus

“…This approach is an appealing alternative to existing iPSC technology because it enables the production of patient-specific NSCs without passing through the pluripotent stage, thereby decreasing the tumorigenic risk (15,16). Since the first mouse iNSCs (miNSCs) were established in 2011 (12), many studies have been published detailing the derivation of iNSCs from a variety of species, including rats (17), monkeys (18), and humans (18)(19)(20). Established iNSCs have numerous features in common with embryonic brain-derived NSCs, including morphology, self-renewal capacity, gene and protein expression profiles, epigenetic state, as well as functional multipotency in vitro and in vivo (9,21,22).…”