Functional Comparison of Blood-Derived Human Neural Progenitor Cells.

Eszter Szabó,János M Réthelyi,Ágota Apáti,László Homolya,Edit Hathy,Zsuzsa Erdei,Flóra Juhász,Dóra Reé

doi:10.3390/ijms21239118

Abstract

Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although the establishment, stabilization, and directed differentiation of iPSC lines are rather expensive and time-demanding. However, interrupted reprogramming by omitting the stabilization of iPSCs may allow for the generation of a plastic stage of the cells and thus provide a shortcut to derive NPSCs directly from tissue samples. Here, we demonstrate a method to generate shortcut NPCs (sNPCs) from blood mononuclear cells and present a detailed comparison of these sNPCs with NPCs obtained from the same blood samples through stable iPSC clones and a subsequent neural differentiation (classical NPCs—cNPCs). Peripheral blood cells were obtained from a schizophrenia patient and his two healthy parents (a case–parent trio), while a further umbilical cord blood sample was obtained from the cord of a healthy new-born. The expression of stage-specific markers in sNPCs and cNPCs were compared both at the protein and RNA levels. We also performed functional tests to investigate Wnt and glutamate signaling and the oxidative stress, as these pathways have been suggested to play important roles in the pathophysiology of schizophrenia. We found similar responses in the two types of NPCs, suggesting that the shortcut procedure provides sNPCs, allowing an efficient screening of disease-related phenotypes.

Highlights

Human neuronal cell cultures are essential for biological and preclinical studies of our nervous system
neural progenitor cells (NPCs) were generated from peripheral blood mononuclear cells (PBMCs) and umbilical cord blood (UCB) and represent three healthy and one diseased donor with a wide variety of ages (59 years to new-born) and balanced sex distribution
Mononuclear cells were separated from peripheral blood or umbilical cord blood and the samples were transduced by Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc

Summary

Introduction

Human neuronal cell cultures are essential for biological and preclinical studies of our nervous system. A direct conversion of fibroblasts or human adult peripheral blood T cells [2] to neuronal cells can be achieved using the overexpression of neuronal transcription factors In this approach, first Achaete-scute homolog 1 (Ascl1), Brain (Brn2), and myelin transcription factor (Myt1l) were used [3], and a wide variety of factors and molecules were applied (reviewed in [4]) to produce induced neurons (iN) in a simpler and shorter process (taking only weeks). Neural progenitor cell (NPC) cultures were established from fibroblasts or blood progenitor cells by starting the reprogramming protocol with delivery of classical Yamanaka factors, using nonintegrating methods. Thereafter, before reaching a pluripotent state, the cells were further differentiated by changing the culture conditions into neurogenic conditions (CASD), with or without growth factor supplementation [6] These methods provided homogeneous and proliferative NPC populations, and the devoted cost and time decreased dramatically by bypassing the iPSC derivation process. For the generation of NPCs, we used the same blood samples and the same Sendai virus transduction for reprogramming and performed comparative studies in terms of the RNA and protein expression profiles and various functional assays

Methods

Results

Conclusion