Increased Telomere Length in Natural Killer Cells Generated from Human Induced Pluripotent Stem Cells

Telomeres are repetitive DNA protein structures that cap the ends of chromosomes, protect chromosome ends from degradation and fusion, and are essential for maintenance of genomic integrity. Telomere length has been shown to gradually shorten over time as cells divide. When telomeres become critically short, the cells enter a state of senescence; as such, telomere shortening has been implicated in accelerated aging. Natural Killer (NK) cells function as key effector cells of the innate immune system. NK cells have significant cytotoxic activity and are responsible for localized expression of pro-inflammatory cytokines; therefore they play an important role in host defense against malignancy and viral infection. NK cells represent a useful tool for cellular therapy due to their ability to kill target cells in a human leukocyte antigen (HLA) non-restricted manner without prior sensitization. Our lab has pioneered methods for the production of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These hESC and iPSC-derived NK cells effectively kill diverse human tumor cells both in vitro and in vivo. One advantage of NK cells from IPSC/hESC sources is the ability to generate a supply of a homogenous NK cell therapy product compared to NK cells isolated from peripheral blood. However, current limitations in therapeutic use of NK cells includes the persistence and expansion of transplanted NK cells in vivo. In order to overcome these barriers, various means for the ex-vivo expansion of large numbers of NK cells prior to administration to patients have been devised. One such method involves propagation of NK cells generated from hESC/iPSC sources with artificial antigen presenting cells (aAPCs). However, aAPC-mediated expansion may lead to telomere shortening. Therefore, aAPCs have been engineered to express either membrane bound IL-15 (mbIL15) or membrane bound IL-21 (mbIL21), which promotes proliferation of NK cells, to ameliorate this effect. We have performed initial studies using real time PCR to measure the telomere length of NK cells generated from iPSC sources and expanded with aAPCs expressing mbIL21 and compared them to aAPC expanded NK cells isolated from peripheral blood. Telomere length in NK cells derived from IPSCs is much longer (200-300kb/diploid genome), compared to those expanded from peripheral blood (<50kb/diploid genome). We will be measuring telomere length and telomerase activity in NK cells expanded using aAPCs expressing mbIL15. In order to better characterize the part that telomeres play in NK cell development, we have also generated iPS cells from fibroblasts of telomerase deficient patients which have mutations in the TERC RNA component. We have demonstrated that these cells appropriately express markers of pluripotency: Oct4, Sox2, SSEA4, and Nanog. We have also demonstrated that these telomerase deficient iPSCs can be differentiated into hematopoietic precursors. The reprogramming of fibroblasts to the pluripotent state results in initial lengthening of telomeres. Telomerase deficient fibroblasts have telomere lengths of 130-150kb/diploid genome compared to normal human fibroblasts with telomeres of ~250kb/diploid genome. After reprogramming, the iPSCs generated from telomerase deficient fibroblasts can have markedly increased telomere lengths to as high as 2000kb/diploid genome. We are currently generating NK cells from these telomerase-deficient iPSCs. We predict that NK cells with longer telomeres from hESC/iPSC sources will persist longer post-transplantation and will be more effective at killing tumor targets and therefore will be superior to those expanded from NK cells from the peripheral blood. We will determine their effectiveness and persistence of these NK cells using in vivo xenograft mouse models. The goal of these studies is to better understand the role of telomerase in lymphocyte development and define methods to better expand or maintain telomeres in therapeutic NK cell-based therapy.