From: Kyunghee, Choi, et al., 1998. A Common Precursor for Hematopoietic and Endothelial Cells . Development 125, pp725-732.
Introduction:
One of the problems in identifying and characterizing precursor stem cells is to access embryos prior to the establishment of blood islands due to the small number of cells at this early stage of development. This paper shows that embryonic stem cells can be differentiated into hematopoietic and endothelial cell lineages in a liquid culture system.
Materials & Methods:
Cell Culture
• Embryonic stem cells were maintained on STO feeder cells or gelatinized flasks and blast cells generated as described by Kennedy et al, 1997.
• Blast colonies were grown in the presence of either vascular endothelial growth factor (VEGF) or c-kit ligand and conditioned medium from an endothelial cell line.
• For generating endothelial cells, individual blast colonies were cultured in microtiter wells containing IMDM with 10% fetal calf serum, 10% horse serum with the following growth factors: vascular endothelial growth factor, insulin growth factor-1, erythropoietin, bF growth factor, interleukin-11, c-kit ligand conditioned medium, endothelial cell growth supplement, L-glutamine and MTG. |
|
A specially collected and processed serum developed exclusively for the promotion of stem cells.
Proprietary formulation developed over 2 years; Collected and processed by an ISO9001-2000 company
Call for more information:
903-592-1363 |
• Following 3-4 days in culture the non-adherent hematopoietic cells were removed and used directly for gene expression studies or assayed for precursors in methyl cellulose containing 10% plasma-derived serum (from Animal Technologies, Inc., Tyler, Texas) , 5% protein free hydridoma medium together with the following cytokines: KL, IL-3, IL-11, CM-DSF, Epo, M-CSF, G-CSF, IL-6, LIF, VEGF.
• Following removal of the non-adherent populations, the adherent cells were cultured for an additional 1-2 weeks in IMDM with 10% fetal calf serum, 10% horse serum, VEGF, IGF-1, bFGF, endothelial cell growth supplement, L-glutamine, and MTG.
• Cells were then harvested by trypsinization for gene expression.
Cell Staining
• Blast colonies were picked onto cover slips coated with a thin layer of matrigel and cultured in 35 mm Petri dishes in medium containing both hematopoietic and endothelial cytokines described above.
• After 4-7 days, the cultures of non-adherent cells were harvested and the cells cultured for an additional 1-2 weeks in medium without hematopoietic cytokines.
• For fluorescence analysis adherent cells were initially cultured in the presence of Dil-Ac-DI. For 2 hours. Following incubation, cells were washed 3 times and fixed for 10 minutes in PBS containing 3% paraformaldehyde and 3% sucrose.
• The fixed cells were washed 2-3 times, incubated initially with Biotin a -mouse CD31 for 1 hour, washed 5 times and then incubated with Streptavidin-FITC for another hour.
• Following the second staining step the cells were washed again 5 times and the overslip with the cells was mounted onto a slide for analysis.
• Images were acquired with a digital confocal microscope and images deconvolved using a nearest neighbor algorithm.
PCR Analysis
RNA isolation and PCR were performed as described by Chomczynski & Sacchi, 1987; and Chelly et al., 1998) See text of paper (Choi, K. et al, (1998)) for details.
TEM Analysis
Adherent cells were processed as described by McDowell and Trump(1976), fixed in s-collidine buffer with 1% Osmium tetroxide for 1 hour stained with uranyl acetate, dehydrated and embedded in polybed 812. Thin sections were cut, stained with uranyl acetate and lead citrate and examined using a JEOL EX 1200 electron microscope.
Results:
This study demonstrated that blast cell colonies generated from embryonic stem cell-derived embryoid bodies contain both hematopoietic and endothelial cell precursors. The demonstration of endothelial precursors in the blast colonies suggests that the blast colony-forming cell (BL-CFU) represent the hemangioblast.
Selected References from Choi, K et al. (1998)
Chelly, J., et al., (1988). Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature 333, 858-860.
Chomczynski, P and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol= chloroform extraction. Analyt. Biochem 162, 156-159.
Kennedy, M., et al. (1997). A common precursor for primitive erythropoisis and definitive hematopoiesis. Nature , 386, 488-493.
McDowell, E.M. and Trump, B.F.H. (1976). Histologic fixative suitable for diagnostic light and electron microscopy. Arch. Path. Lab. Med. 100, 405-414.
James Koukl, Ph.D.
Scientific Consultant
Animal Technologies, Inc.
Tyler, Texas
|
