Get with the program

DURHAM, N.C.—Stem cells attract a significant amount ofinterest in the industry for their potential in a variety of fields, especiallyregenerative medicine. The current leading method for reprogramming inducedpluripotent stem cells (iPSCs) consists of combining iPSCs with mouse or humanstromal cells during differentiation, but this approach runs the risk offoreign DNA being introduced to the reprogrammed cells.

Researchers from the Department of Medicine and Institutefor Human Genetics at University of California, San Francisco, however, havedeveloped a new method that stands to offer purer, safer stem cells that arepatient-specific and lack the risk of rejection and damage by alien DNA. Thework was led by Dr. Yuet Wai Kan and Dr. Lin Ye.

The new study focused on CD34+ cells, a type of blood stemcell that has been linked to proliferation. The issue with this class of cellsis that in order to collect enough of them from a patient to produce bloodrequires a large amount of blood to be collected.

This new method makes use of Sendai viral vectors togenerate iPSCs "from adult mobilized CD34+ and peripheral blood mononuclearcells (MNC)," said Kan. "Sendai virus is an RNA virus that carries no risk ofaltering the host genome, so is considered an efficient solution for generatingsafe iPSC."

"Just 2 milliliters of blood yielded iPS cells from whichhematopoietic stem and progenitor cells could be generated. These cells couldcontain up to 40 percent CD34+ cells, of which approximately 25 percent werethe type of precursors that could be differentiated into mature blood cells.These interesting findings reveal a protocol for the generation iPSCs using areadily available cell type," said Ye. "We also found that MNCs can beefficiently reprogrammed into iPSCs as readily as CD34+ cells. Furthermore,these MNCs derived iPSCs can be terminally differentiated into mature bloodcells."

An MNC is any blood cell that features a round nucleus, andcan include lymphocytes, monocytes or macrophages.

"Currently, there are only two no-integrating methods thathave been used to generate iPSCs: the EBNA1/OriP plasmid and Sendai viralvector. The EBNA1/OriP plasmid method requires a large amount of blood toprepare enough MNCs (1–2X106) for reprogramming. Sendai viral vectors need manyfewer blood cells (2–3X104)," Ye explains. "In this study, MNCs prepared from 2ml of blood are more than enough to generate functional iPSCs. In addition,with the EBNA1/OriP plasmid DNA vectors, as they are introduced into thenucleus, occasional integration into the host genome cannot be ruled out. Incontrast, Sendai viral vectors-mediated reprogramming is efficient, and as anRNA virus that only replicates in cytoplasm, it does not enter the nucleus andtherefore will not integrate into the host genome."

Sendai viral vectors generate iPSCs through theover-expression of four transcription factors (OCT4, SOX2, KLF4 and cMYC) insomatic cells and inducing somatic cell reprogramming, says Ye. When Sendaiviral vectors are manipulated to carry the four transcription factors, they caninfect somatic cells to induce reprogramming. And since the Sendai viral genome"has been altered to carry temperature sensitive mutations, the virus could beremoved when cells divide," he adds.

"The development of iPSC technology opens up a new avenuefor the field of personalized regenerative medicine since it is possible togenerate patient-specific pluripotent stem cells for modeling disease and fordeveloping cell therapies," says Ye.

He notes that their laboratory has begun examining the possibilityof using this technology to treat sickle cell anemia and thalassemia, two blooddisorders in which the body produces abnormal red blood cells or hemoglobin. Insickle cell anemia, a mutation in the hemoglobin gene causes the body toproduce red blood cells shaped like a sickle, rather than a disc, which canhamper blood flow. In thalassemia, an abnormal form of hemoglobin is producedthat leads to damage and destruction of red blood cells, resulting in anemia.

"Our laboratory has been interested in studying sickle cellanemia and thalassemia for some years and is now exploring the feasibility ofusing the iPSC technology to treat these diseases," says Ye. "The proposedapproach is to generate iPSCs from these patients, correct the b-globin-gene mutationsand differentiate the iPSCs to hematopoietic cells for autotransplantation. Wewill conduct gene correction in iPSCs as well as in-vivo study to find out whether the hematopoietic progenitorcells differentiated from iPSCs could be engrafted or not."

The results of this study were published in STEM CELLS Translational Medicine. Thepaper, "Blood cell-derived induced pluripotent stem cells free of reprogrammingfactors generated by Sendai viral vectors," first appeared online July 11.