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Stem Cell Res Ther. 2019 Mar 12;10(1):83. doi: 10.1186/s13287-019-1163-7.

A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors.

Author information

1
Department of Anesthesiology, University of California San Diego School of Medicine, La Jolla, CA, 92093, USA.
2
Department of Histology and Embryology, Faculty of Medicine, Masaryk University Brno, Kamenice 3, 62500, Brno, Czech Republic.
3
Gene Expression Laboratory, Howard Hughes Medical Institute and Salk Institute for Biological Studies, 10010 North Torrey Pines Rd, La Jolla, CA, 92037, USA.
4
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, 92093, USA.
5
Institute of Animal Physiology and Genetics, v.v.i., AS CR, Liběchov, Czech Republic.
6
Primary and Stem Cell Systems, Life Technologies (Thermo Fisher Scientific), 501 Charmany Drive, Madison, WI, 53719, USA.
7
Sanford Stem Cell Clinical Center, University of California San Diego, La Jolla, CA, 92093, USA.
8
Gene Expression Laboratory, Howard Hughes Medical Institute and Salk Institute for Biological Studies, 10010 North Torrey Pines Rd, La Jolla, CA, 92037, USA. pfaff@salk.edu.
9
Department of Anesthesiology, University of California San Diego School of Medicine, La Jolla, CA, 92093, USA. mmarsala@ucsd.edu.
10
Sanford Consortium for Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA, 92037, USA. mmarsala@ucsd.edu.

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 (SOD1G93A), 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.

KEYWORDS:

Amyotrophic lateral sclerosis (ALS); Bioinformatic tools to study xenografts; Human embryonic stem cell (hESC); Neural stem cell (NSC); Spinal cord; Spinal traumatic injury

PMID:
30867054
DOI:
10.1186/s13287-019-1163-7
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