Direct isolation of human central nervous system stem cells

N Uchida

D Buck

D He

M Reitsma

^{StemCells, Inc., 525 Del Rey Avenue, Suite C, Sunnyvale, CA 94085;}^{Laboratory of Genetics, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037; and}^{Departments of Pathology and Developmental Biology, Stanford University, Stanford, CA 90305}

^{To whom reprint requests should be addressed. E-mail:}ten.llecmets@adihcun.

Contributed by Irving L. Weissman

Accepted 2000 Oct 20.

Abstract

Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These hCNS-SC are phenotypically 5F3 (CD133)^{, 5E12}^{, CD34}^{, CD45}^{, and CD24}^{. Single CD133}^CD34^CD45^{sorted cells initiated neurosphere cultures, and the progeny of clonogenic cells could differentiate into both neurons and glial cells. Single cells from neurosphere cultures initiated from CD133}^CD34^CD45^{cells were again replated as single cells and were able to reestablish neurosphere cultures, demonstrating the self-renewal potential of this highly enriched population. Upon transplantation into brains of immunodeficient neonatal mice, the sorted/expanded hCNS-SC showed potent engraftment, proliferation, migration, and neural differentiation.}

Keywords: neural, transplantation, clonogenic, self-renewal, differentiation

Abstract

Although much is known about the development of the brain during embryonic life, the pathways and lineages that neural precursors take in neurogenesis during development and adult life are only partly understood (1). Until recently, it was unclear whether neurogenesis proceeded like hematopoiesis, i.e., from pluripotent clonogenic stem cells that both self-renew to produce multipotent progenitors and give rise to progressively more committed progeny (2), or if some other developmental hierarchy, not including stem cells, was operative. Recently, long-term cell culture systems have been developed for rodent and human central nervous system (CNS) cells that continuously propagate a heterogeneous population of early neural stem and/or progenitor cells. CNS neural stem/progenitor cells can grow either in monolayers on substrate-coated tissue culture plates (3, 4) or as self-adherent complexes of cells, forming clusters known as neurospheres (5, 6). In both culture systems, these cells maintain the capacity to produce the three main mature cell classes of the CNS: neurons, astrocytes, and oligodendrocytes (6, 7). In vivo analysis of transplanted cultured rodent cells shows engraftment, migration, and site-specific multilineage differentiation in mice and rats (8, 9). Human long-term cultured neurosphere cells show similar engraftment, migration, and site-specific differentiation upon transplantation into immunosuppressed rats (10). These rodent and human studies suggest that the neural cultures support the growth of stem cells and/or progenitors capable of engraftment and differentiation (3–14). Recently, Roy et al. (13) showed that human primitive progenitor cells from the dentate gyrus of adult hippocampus can be selected by transfecting them with the reporter green fluorescent protein gene driven by the nestin enhancer. However, the direct isolation of human neural stem cells from fresh tissues through the identification of cell surface markers and fluorescence-activated cell sorting (FACS) of either the central or peripheral nervous system, to our knowledge, has not yet been reported.

In the past decade, hematopoietic stem and progenitor cells have been identified by using mAbs against cell surface markers for enriching rare subpopulations that are clonal self-renewing and multipotent stem cells (15, 16) or oligopotent progenitors (17, 18). This strategy was used successfully to isolate rat peripheral nervous system stem cells (14) and, as we report here, to identify and isolate a candidate human CNS stem cell (hCNS-SC). The mAb 5F3 recognizes the CD133 antigen.^¶ Antibodies to CD133 define a five-transmembrane protein and have been used to enrich for human hematopoietic stem cells (19). Here we show that mAbs 5F3 and the novel mAb, 5E12, detect a distinct subset of human fetal brain (FBr) cells. FACS using these mAbs results in a subset of human CD133^{FBr cells that are capable of neurosphere initiation, self-renewal, and multilineage differentiation at the single-cell level. Another mAb, 8G1, which recognizes human CD24, can further enrich neurosphere initiating cell activity within the CD133}^CD24^{fraction. Because the CD133}^{cells self-renew, significantly expand in neurosphere cultures, and differentiate}in vitro to neurons and glial cells, they are candidate human CNS-SC. Moreover, transplantation of CD133^{sorted/expanded neurosphere cells into the lateral ventricles of newborn nonobese diabetic–severe combined immunodeficient (NOD-SCID) mouse brains resulted in specific engraftment in numerous sites of the brain, which is similar to results shown by Fricker}et al. (10). These cells also demonstrated continued self-renewal, migration, and neural differentiation for at least 7 months.

Limiting dilution assays on the above cell populations were performed and NS-IC frequencies were determined by linear regression analysis.

Acknowledgments

We are grateful to David Anderson, Ben Barres, and Eric Lagasse for stimulating discussions and advice; M. L. Gage for critical review of the manuscript; and Linda Kitabayashi for performing confocal microscopic analysis. We also thank the staff of StemCells, Inc., for their continuous encouragement and support.

Acknowledgments

Abbreviations

CNS	central nervous system
hCNS-SC	human CNS stem cell
FACS	fluorescence-activated cell sorting
FBr	fetal brain
NOD-SCID	nonobese diabetic–severe combined immunodeficient
FSC	fetal spinal cord
NS-IC	neurosphere-initiating cells
GFAP	glial fibrillary acidic protein
N-CAM	neural cell adhesion molecule
SVZ	subventricular zone
RMS	rostral migratory stream

Abbreviations

Footnotes

^{7th Workshop and Conference on Human Leucocyte Differentiation Antigens, Harrogate, U.K., June 20–24, 2000.}

Footnotes

References

1. Gage F H. Science. 2000;287:1433–1438.[PubMed]
2. Weissman I L. Cell. 2000;100:157–168.[PubMed]
3. Ray J, Peterson D A, Schinstine M, Gage F H. Proc Natl Acad Sci USA. 1993;90:3602–3606.
4. McKay R. Science. 1997;276:66–71.[PubMed]
5. Reynolds B A, Tetzlaff W, Weiss S. J Neurosci. 1992;12:4565–4574.
6. Weiss S, Reynolds B A, Vescovi A L, Morshead C, Craig C G, van der Kooy D. Trends Neurosci. 1996;19:387–393.[PubMed]
7. Carpenter M K, Cui X, Hu Z Y, Jackson J, Sherman S, Seiger A, Wahlberg L U. Exp Neurol. 1999;158:265–278.[PubMed]
8. Lois C, Alvarez-Buylla A. Science. 1994;264:1145–1148.[PubMed]
9. Suhonen J O, Peterson D A, Ray J, Gage F H. Nature (London) 1996;383:624–627.[PubMed]
10. Fricker R A, Carpenter M K, Winkler C, Greco C, Gates M A, Bjorklund A. J Neurosci. 1999;19:5990–6005.
11. Flax J D, Aurora S, Yang C, Simonin C, Wills A M, Billinghurst L L, Jendoubi M, Sidman R L, Wolfe J H, Kim S U, et al Nat Biotechnol. 1998;16:1033–1039.[PubMed][Google Scholar]
12. Brustle O, Choudhary K, Karram K, Huttner A, Murray K, Dubois-Dalcq M, McKay R D. Nat Biotechnol. 1998;16:1040–1044.[PubMed]
13. Roy N S, Wang S, Jiang L, Kang J, Benraiss A, Harrison-Restelli C, Fraser R A, Couldwell W T, Kawaguchi A, Okano H, et al Nat Med. 2000;6:271–277.[PubMed][Google Scholar]
14. Morrison S J, White P M, Zock C, Anderson D J. Cell. 1999;96:737–749.[PubMed]
15. Spangrude G J, Heimfeld S, Weissman I L. Science. 1988;241:58–62.[PubMed]
16. Morrison S J, Weissman I L. Immunity. 1994;1:661–673.[PubMed]
17. Kondo M, Weissman I L, Akashi K. Cell. 1997;91:661–672.[PubMed]
18. Akashi K, Traver D, Miyamoto T, Weissman I L. Nature (London) 2000;404:193–197.[PubMed]
19. Yin A H, Miraglia S, Zanjani E D, Almeida-Porada G, Ogawa M, Leary A G, Olweus J, Kearney J, Buck D W. Blood. 1997;90:5002–5012.[PubMed]
20. Lefkovits I, Waldmann H Limiting Dilution Analysis of Cells of the Immune System. Oxford: Oxford Univ. Press; 1999. [[PubMed][Google Scholar]
21. Baum C M, Weissman I L, Tsukamoto A S, Buckle A M, Peault B. Proc Natl Acad Sci USA. 1992;89:2804–2808.
22. Uchida N, Weissman I L. J Exp Med. 1992;175:175–184.
23. Miraglia S, Godfrey W, Yin A H, Atkins K, Warnke R, Holden J T, Bray R A, Waller E K, Buck D W. Blood. 1997;90:5013–5021.[PubMed]
24. Corbeil D, Roper K, Hellwig A, Tavian M, Miraglia S, Watt S M, Simmons P J, Peault B, Buck D W, Huttner W B. J Biol Chem. 2000;275:5512–5520.[PubMed]
25. Uchida N, Combs J, Chen S, Zanjani E, Hoffman R, Tsukamoto A. Blood. 1996;88:1297–1305.[PubMed]
26. Visser J W, Bauman J G, Mulder A H, Eliason J F, de Leeuw A M. J Exp Med. 1984;159:1576–1590.
27. Schluter C, Duchrow M, Wohlenberg C, Becker M H, Key G, Flad H D, Gerdes J. J Cell Biol. 1993;123:513–522.
28. Gage F H, Cristen Y Isolation, Characterization and Utilization of CNS Stem Cells. Heidelberg: Springer; 1997. [PubMed][Google Scholar]
29. Gage F H, Coates P W, Palmer T D, Kuhn H G, Fisher L J, Suhonen J O, Peterson D A, Suhr S T, Ray J. Proc Natl Acad Sci USA. 1995;92:11879–11883.
30. Palmer T D, Takahashi J, Gage F H. Mol Cell Neurosci. 1997;8:389–404.[PubMed]
31. Yandava B D, Billinghurst L L, Snyder E Y. Proc Natl Acad Sci USA. 1999;96:7029–7034.
32. Armstrong R J, Svendsen C N. Cell Transplant. 2000;9:139–152.[PubMed]
33. Brustle O, Jones K N, Learish R D, Karram K, Choudhary K, Wiestler O D, Duncan I D, McKay R D. Science. 1999;285:754–756.[PubMed]
34. McDonald J W, Liu X Z, Qu Y, Liu S, Mickey S K, Turetsky D, Gottlieb D I, Choi D W. Nat Med. 1999;5:1410–1412.[PubMed]
35. Lee S H, Lumelsky N, Studer L, Auerbach J M, McKay R D. Nat Biotechnol. 2000;18:675–679.[PubMed]
36. Johansson C B, Momma S, Clarke D L, Risling M, Lendahl U, Frisen J. Cell. 1999;96:25–34.[PubMed]
37. Barres B A. Cell. 1999;97:667–670.[PubMed]
38. Clarke D L, Johansson C B, Wilbertz J, Veress B, Nilsson E, Karlstrom H, Lendahl U, Frisen J. Science. 2000;288:1660–1663.[PubMed]
39. Doetsch F, Caille I, Lim D A, Garcia-Verdugo J M, Alvarez-Buylla A. Cell. 1999;97:703–716.[PubMed]
40. Chiasson B J, Tropepe V, Morshead C M, van der Kooy D. J Neurosci. 1999;19:4462–4471.
41. Trojanowski J Q, Mantione J R, Lee J H, Seid D P, You T, Inge L J, Lee V M. Exp Neurol. 1993;122:283–294.[PubMed]
42. Auerbach J M, Eiden M V, McKay R D. Eur J Neurosci. 2000;12:1696–1704.[PubMed]