Cell-based therapies against HIV/AIDS have been gaining increased interest. NK cell
Cell-based therapies against HIV/AIDS have been gaining increased interest. NK cell function is usually often compromised in HIV-1-infected individuals (14 15 Additionally while CYN-154806 the treatment of HIV-1-infected individuals with antiretroviral medications effectively reduces viral loads the restoration of cellular immunity in treated patients proceeds slowly and they may never return to their preinfection status (15 25 Cell-based immunotherapy using either T or CYN-154806 NK effector cells has been used to treat malignancies such as leukemia melanoma and renal cell carcinoma (35 39 40 KIAA1732 Comparable strategies have also been postulated as a novel therapeutic approach for HIV-1 treatment CYN-154806 in clinical practice (1 24 One notable case exhibited an apparent remedy of HIV-1 contamination by hematopoietic cell transplantation using a donor deficient for the expression of the HIV-1 coreceptor CCR5 (24). However this patient was transplanted primarily to treat acute myelogenous leukemia and a wider application of this strategy is severely limited due to high risk associated with transplantation and low availability of suitable HLA-matched CCR5-deficient donors. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide potential alternative approaches to generating hematopoietic cells for therapy against HIV. Our group as well as others have reported that hESCs and iPSCs can give rise to diverse lymphoid and myeloid lineages (3 11 18 42 46 50 51 Additional studies have exhibited that hESC-derived macrophages and dendritic cells are susceptible to HIV-1 contamination indicating the potential of hESCs for HIV cell/gene therapy (3 5 Previously our group exhibited the derivation of NK cells from hESCs with the ability to potently kill multiple types of tumor cells both and (50 51 We now demonstrate the successful derivation of NK cells from human induced pluripotent stem cells (iPSCs). Additionally we find that these hESC- and iPSC-derived NK cells have potent anti-HIV-1 activity. Therefore these studies establish the use of hESC- and iPSC-derived NK cells as a novel system to better understand anti-HIV immunity and suggest the potential to establish a readily available cell-based strategy to treat HIV/AIDS. MATERIALS AND METHODS NK cell differentiation from hESCs and iPSCs. The hESC line H9 (Wicell Madison WI) and iPSC CYN-154806 line BJ1-iPS12 (36) (kindly provided by George Daley Boston Children’s Hospital) were maintained as undifferentiated cells as described previously (29). A two-step stromal cell coculture system was used for NK cell differentiation from hESCs and iPSCs as described previously (50 51 Briefly hESCs or iPSCs were cocultured with the murine bone marrow stromal cell line M210-B4 (American Type Culture Collection Manassas VA) for 19 to 21 days to allow hematopoietic differentiation. CD34+CD45+ hematopoietic progenitors were then enriched by using EasySep selection kits (Stem Cell Technologies) and cocultured with a confluent monolayer of irradiated murine AFT024 cells (fetal liver-derived stromal cell line; kindly provided by K. Moore and I. Lemischka) under NK cell culture conditions for 4 to 5 weeks. Cells were harvested and analyzed for phenotype and function. UCB-NK cells derived CYN-154806 under the same conditions were used as controls in all assays. Phenotyping of hESC- and iPSC-derived NK cells. Single-cell suspensions were stained with allophycocyanin (APC)- phycoerythrin (PE)- fluorescein isothiocyanate (FITC)- and peridinin chlorophyll protein (PerCP)-cy5.5-coupled IgG or specific antibodies against human blood surface antigens including CD45-PE CD56-APC CD16-PerCP-cy5.5 NKG2D-PE NKp44-PE NKp46-PE TRAIL-PE CD158b-FITC CD158e1/2-FITC (all from BD Pharmingen) FasL-PE (R&D) CD158a/h-PE and CD158i-PE (Beckman Coulter) as shown in Fig. ?Fig.1.1. All analyses were performed with a FACSCalibur instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star). NK cells isolated from peripheral blood (PB-NK) using an NK cell-negative selection kit (Miltenyi Biotech) were used as positive controls. FIG. 1. Hematopoietic development from hESCs and iPSCs cocultured with the stromal cell line M210-B4. hESCs and iPSCs were first allowed to differentiate on M210-B4 stromal cells for 19 to 21 days to develop hematopoietic.