Chicken embryonic brain: an in vivo model for verifying neural stem cell potency

Laboratory investigation

Alex Kharazi M.D., Ph.D.1, Michael L. Levy M.D., Ph.D.2,3, Maria Cristina Visperas B.S.1,4,5, and Chih-Min Lin Ph.D.1,6
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  • 1 Department of Research and Development, Stemedica Cell Technologies, Inc., San Diego;
  • | 2 Division of Pediatric Neurosurgery, Rady Children's Hospital San Diego;
  • | 3 Division of Neurosurgery;
  • | 4 Science Studies Program, and
  • | 5 Department of Communication, University of California, San Diego; and
  • | 6 Department of Research and Development, Animal Cell Therapies Inc., La Jolla, California
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Object

The multipotency of neural stem cells (NSCs) can be assessed in vitro by detection of stage-specific markers in response to a suitable differentiation signal. This test is frequently used because it is fast and affordable. However, it is not clear how the in vitro potential for multilineage differentiation and stem cell marker expression would reflect the ability of NSCs to engraft into the brain following transplantation. The authors undertook this study to directly compare the in vitro potency and in vivo migration of human NSCs (hNSCs) expanded under conditions of gradually increased concentration of fetal bovine serum (FBS) as a maturation factor.

Methods

Human NSCs isolated from fetal brain were propagated in serum free media (SF-hNSCs) and in media containing 0.1% and 0.2% serum. At Passage 4 in tissue culture the NSCs were harvested and either differentiated in vitro or transplanted into the lateral ventricle of chicken embryonic brain at the late stage of its development (Hamburger and Hamilton Stage 26). The in vitro differentiation was evaluated by immunostaining with neural or glial specific markers, and the in vivo migration was assessed using immunohistology.

Results

The authors found that SF-hNSCs successfully engrafted into the chicken embryonic brain, which correlated with their ability to differentiate in vitro. NSCs grown at as low as 0.1% concentration of FBS failed to demonstrate the robust in vivo migration pattern but still preserved the capability to differentiate in vitro. Furthermore, NSCs generated in media containing a higher concentration of FBS (0.2%) lost both the in vivo engraftment and in vitro differentiation potential.

Conclusions

The present study suggests that marker expression and in vitro differentiation assays might not provide adequate information regarding the behavior of NSCs following their transplantation. The in vivo migration following injection into chicken embryonic brain may provide an important assay of the potency of NSCs.

Abbreviations used in this paper:

FBS = fetal bovine serum; GFAP = glial fibrillary acidic protein; HH26 = Hamburger and Hamilton Stage 26; hNSC = human NSC; NSC = neural stem cell; OCT = optimal cutting temperature; PBS = phosphate-buffered saline; SF-hNSC = hNSC grown in a serum-free environment; TUJ1 = tubulin β-III (neuronal Class III β-tubulin).

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