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.
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.
Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA 92037, USA.
O
O
Kakinohana
J
J
Juhasova
S
S
Juhas
J
J
Motlik
O
O
Platoshyn
J
J
Galik
M
M
Hefferan
S H
SH
Yuan
J G
JG
Vidal
C T
CT
Carson
S
S
van Gorp
D
D
Goldberg
M
M
Leerink
P
P
Lazar
S
S
Marsala
A
A
Miyanohara
S
S
Keshavarzi
J D
JD
Ciacci
M
M
Marsala
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D000089662:Doublecortin Protein; D058732:Embryoid Bodies / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D016867:Immunocompromised Host; D019394:Ki-67 Antigen / Q000378:metabolism; D008297:Male; D051379:Mice; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D009474:Neurons / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020760:Spinal Cord Ischemia / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D013552:Swine; D013556:Swine, Miniature; D014157:Transcription Factors / Q000378:metabolism
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C000714847:Dcx protein, mouse; C000714848:Dcx protein, rat; D000089662:Doublecortin Protein; D019394:Ki-67 Antigen; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D014157:Transcription Factors