Improved Efficiency and Pace of Generating Induced Pluripotent Stem Cells from Human Adult and Fetal Fibroblasts

¹ Stem Cell Program, Institute for Cell Engineering, and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205
² Stem Cell Program, Institute for Cell Engineering, and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Graduate Program in Immunology, Johns Hopkins University, Baltimore, MD 21205
³ Stem Cell Program, Institute for Cell Engineering, and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Stem Cell Resources Center, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205
⁴ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205
⁵ Stem Cell Program, Institute for Cell Engineering, and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205
⁶ Stem Cell Program, Institute for Cell Engineering, and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205; Stem Cell Resources Center, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205

^* To whom correspondence should be addressed. E-mail: lcheng{at}welch.jhu.edu.

	Abstract

It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles human embryonic stem (hES) cells. This was achieved by ectopic expression of 4 genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low (10^-4), especially for postnatal human fibroblasts. We have started supplementing an additional gene or bioactive molecules to increase efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the previously used 4 reprogramming genes enhanced the efficiency by 23-70 fold from both human adult and fetal fibroblasts. Discernible hES-like colonies also emerged 1-2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs by immortalized human feeder cells we previously established for optimal hES cell growth. We have further characterized individually picked hES-like colonies after expansion (up to 24 passages so far). The majority of them expressed various undifferentiated hES markers. Some but not all the hES-like clones can be induced to differentiate into the derivatives of the 3 embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or BMP-4 induction as classic hES cells. Using this improved approach, we also generated hES-like cells from homozygous fibroblasts containing the sickle cell anemia mutation HbS.

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