A Scalable Solution for Isolating Human Multipotent Clinical-grade Neural Stem Cells from ES Precursors

Overview

Journal Stem Cell Res Ther

Publisher Biomed Central

Date 2019 Mar 15

PMID 30867054

Citations 24

Authors

Dasa Bohaciakova

Marian Hruska-Plochan

Rachel Tsunemoto

Wesley D Gifford

Shawn P Driscoll

Thomas D Glenn

Stephanie Wu

Silvia Marsala

Michael Navarro

Takahiro Tadokoro

Stefan Juhas

Jana Juhasova

Oleksandr Platoshyn

David Piper

Vickie Sheckler

Dara Ditsworth

Samuel L Pfaff

Martin Marsala

Affiliations

Soon will be listed here.

Abstract

Background: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation.

Methods: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1), or (iii) spinally injured immunosuppressed minipigs.

Results: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics.

Conclusions: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.

Citing Articles

Genomic tumor evolution dictates human medulloblastoma progression.

Ruchiy Y, Tsea I, Preka E, Verhoeven B, Olsen T, Mei S Neurooncol Adv. 2024; 6(1):vdae172.

PMID: 39659836 PMC: 11629688. DOI: 10.1093/noajnl/vdae172.

Human iPSC-derived neural stem cells displaying radial glia signature exhibit long-term safety in mice.

Luciani M, Garsia C, Beretta S, Cifola I, Peano C, Merelli I Nat Commun. 2024; 15(1):9433.

PMID: 39487141 PMC: 11530573. DOI: 10.1038/s41467-024-53613-7.

Stem cell-based ischemic stroke therapy: Novel modifications and clinical challenges.

Sun Y, Jiang X, Gao J Asian J Pharm Sci. 2024; 19(1):100867.

PMID: 38357525 PMC: 10864855. DOI: 10.1016/j.ajps.2023.100867.

A model of human neural networks reveals NPTX2 pathology in ALS and FTLD.

Hruska-Plochan M, Wiersma V, Betz K, Mallona I, Ronchi S, Maniecka Z Nature. 2024; 626(8001):1073-1083.

PMID: 38355792 PMC: 10901740. DOI: 10.1038/s41586-024-07042-7.

Routes and methods of neural stem cells injection in cerebral ischemia.

Yang X, Zhang X, Cao J, Wu M, Chen S, Chen L Ibrain. 2023; 9(3):326-339.

PMID: 37786754 PMC: 10527797. DOI: 10.1002/ibra.12122.

References

Reubinoff B, Itsykson P, Turetsky T, Pera M, Reinhartz E, Itzik A . Neural progenitors from human embryonic stem cells. Nat Biotechnol. 2001; 19(12):1134-40. DOI: 10.1038/nbt1201-1134. View

Hefferan M, Johe K, Hazel T, Feldman E, Lunn J, Marsala M . Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS. Cell Transplant. 2011; 20(8):1153-61. DOI: 10.3727/096368910X564553. View

Tabar V, Panagiotakos G, Greenberg E, Chan B, Sadelain M, Gutin P . Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat Biotechnol. 2005; 23(5):601-6. DOI: 10.1038/nbt1088. View

Crook J, Peura T, Kravets L, Bosman A, Buzzard J, Horne R . The generation of six clinical-grade human embryonic stem cell lines. Cell Stem Cell. 2008; 1(5):490-4. DOI: 10.1016/j.stem.2007.10.004. View

Sundberg M, Jansson L, Ketolainen J, Pihlajamaki H, Suuronen R, Skottman H . CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells. Stem Cell Res. 2009; 2(2):113-24. DOI: 10.1016/j.scr.2008.08.001. View

Kakinohana O, Juhasova J, Juhas S, Motlik J, Platoshyn O, Galik J . Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study. Cell Transplant. 2012; 21(12):2603-19. DOI: 10.3727/096368912X653200. View

Li X, Du Z, Zarnowska E, Pankratz M, Hansen L, Pearce R . Specification of motoneurons from human embryonic stem cells. Nat Biotechnol. 2005; 23(2):215-21. DOI: 10.1038/nbt1063. View

Sevc J, Goldberg D, Van Gorp S, Leerink M, Juhas S, Juhasova J . Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: effect on spinally grafted human neural precursor survival. Exp Neurol. 2013; 248:85-99. DOI: 10.1016/j.expneurol.2013.05.017. View

Casarosa S, Bozzi Y, Conti L . Neural stem cells: ready for therapeutic applications?. Mol Cell Ther. 2015; 2:31. PMC: 4452059. View

10.

Perrier A, Tabar V, Barberi T, Rubio M, Bruses J, Topf N . Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2004; 101(34):12543-8. PMC: 515094. DOI: 10.1073/pnas.0404700101. View

11.

Doerr J, Schwarz M, Wiedermann D, Leinhaas A, Jakobs A, Schloen F . Whole-brain 3D mapping of human neural transplant innervation. Nat Commun. 2017; 8:14162. PMC: 5253698. DOI: 10.1038/ncomms14162. View

12.

Lu P, Wang Y, Graham L, McHale K, Gao M, Wu D . Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell. 2012; 150(6):1264-73. PMC: 3445432. DOI: 10.1016/j.cell.2012.08.020. View

13.

Lukovic D, Diez Lloret A, Stojkovic P, Rodriguez-Martinez D, Perez Arago M, Rodriguez-Jimenez F . Highly Efficient Neural Conversion of Human Pluripotent Stem Cells in Adherent and Animal-Free Conditions. Stem Cells Transl Med. 2017; 6(4):1217-1226. PMC: 5442830. DOI: 10.1002/sctm.16-0371. View

14.

Aoki M, Kato S, Nagai M, Itoyama Y . Development of a rat model of amyotrophic lateral sclerosis expressing a human SOD1 transgene. Neuropathology. 2005; 25(4):365-70. DOI: 10.1111/j.1440-1789.2005.00611.x. View

15.

Pruszak J, Sonntag K, Aung M, Sanchez-Pernaute R, Isacson O . Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells. 2007; 25(9):2257-68. PMC: 2238728. DOI: 10.1634/stemcells.2006-0744. View

16.

Barnabe-Heider F, Goritz C, Sabelstrom H, Takebayashi H, Pfrieger F, Meletis K . Origin of new glial cells in intact and injured adult spinal cord. Cell Stem Cell. 2010; 7(4):470-82. DOI: 10.1016/j.stem.2010.07.014. View

17.

Squillaro T, Alessio N, Di Bernardo G, Ozcan S, Peluso G, Galderisi U . Stem Cells and DNA Repair Capacity: Muse Stem Cells Are Among the Best Performers. Adv Exp Med Biol. 2018; 1103:103-113. DOI: 10.1007/978-4-431-56847-6_5. View

18.

Golebiewska A, Atkinson S, Lako M, Armstrong L . Epigenetic landscaping during hESC differentiation to neural cells. Stem Cells. 2009; 27(6):1298-308. DOI: 10.1002/stem.59. View

19.

Van Gorp S, Leerink M, Kakinohana O, Platoshyn O, Santucci C, Galik J . Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation. Stem Cell Res Ther. 2013; 4(3):57. PMC: 3706882. DOI: 10.1186/scrt209. View

20.

Elkabetz Y, Studer L . Human ESC-derived neural rosettes and neural stem cell progression. Cold Spring Harb Symp Quant Biol. 2009; 73:377-87. DOI: 10.1101/sqb.2008.73.052. View