Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (Delta CD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused Delta CD4 to an intracellular enhanced green fluorescent protein domain (Delta CD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of Delta CD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of Delta CD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of Delta CD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.