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

O. Kakinohana; J. Juhasova; S. Juhas; J. Motlik; O. Platoshyn; J. Galik; M. Hefferan; S. H. Yuan; J. G. Vidal; C. T. Carson; S. Van Gorp; D. Goldberg; M. Leerink; P. Lazar; S. Marsala; A. Miyanohara; S. Keshavarzi; J. D. Ciacci; M. Marsala M.D.

doi:10.3727/096368912X653200

Cell Transplantation (Dec 2012)

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

O. Kakinohana,
J. Juhasova,
S. Juhas,
J. Motlik,
O. Platoshyn,
J. Galik,
M. Hefferan,
S. H. Yuan,
J. G. Vidal,
C. T. Carson,
S. Van Gorp,
D. Goldberg,
M. Leerink,
P. Lazar,
S. Marsala,
A. Miyanohara,
S. Keshavarzi,
J. D. Ciacci,
M. Marsala M.D.

Affiliations

O. Kakinohana: Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA
J. Juhasova: Institute of Animal Physiology and Genetics, AS CR, Liběchov, Czech Republic
S. Juhas: Institute of Animal Physiology and Genetics, AS CR, Liběchov, Czech Republic
J. Motlik: Institute of Animal Physiology and Genetics, AS CR, Liběchov, Czech Republic
O. Platoshyn: Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA
J. Galik: Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Safarik University, Košice, Slovakia
M. Hefferan: Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA
S. H. Yuan: Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA, USA
J. G. Vidal: BD Biosciences, La Jolla, CA, USA
C. T. Carson: BD Biosciences, La Jolla, CA, USA
S. Van Gorp: Department of Anesthesiology, School for Mental Health and Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands
D. Goldberg: Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA
M. Leerink: Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA
P. Lazar: University of Veterinary Medicine and Pharmacy, Department of Breeding and Diseases of Game and Fish, Košice, Slovakia
S. Marsala: Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA
A. Miyanohara: Vector Core Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA
S. Keshavarzi: Division of Neurosurgery, University of California, San Diego, La Jolla, CA, USA
J. D. Ciacci: Division of Neurosurgery, University of California, San Diego, La Jolla, CA, USA
M. Marsala M.D.: Institute of Neurobiology, Slovak Academy of Sciences, Košice, Slovakia

DOI: https://doi.org/10.3727/096368912X653200
Journal volume & issue: Vol. 21

Abstract

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In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184 + /CD271 - /CD44 - /CD24 + population of ES-hNPCs FAC-sorted and expanded. Male Sprague–Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen–Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2–L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2–4 weeks after grafting, double-labeled hNUMA/ DCX-immunoreactive neurons were seen with extensive DCX + processes. At survival intervals of 4–8 weeks, hNSE + neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA + cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2–6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

Published in Cell Transplantation

ISSN: 0963-6897 (Print); 1555-3892 (Online)
Publisher: SAGE Publishing
Country of publisher: United States
LCC subjects: Medicine
Website: http://journals.sagepub.com/home/cll

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