Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34⁺progenitor cells

Abstract

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specifi c lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/ NKG2A-negative cells generated under different culture conditions showed strong and specifi c cytolytic activity which could have impact on further immunotherapeutic strategies.

PECULIARITIES OF NK CELLS DIFFERENTIATION: CD56dim AND CD56bright NK CELLS AT PREGNANCY AND IN NON-PREGNANT STATE

“…High expression of EGR-2, ID-2, T-bet, and Gata-3 factors is connected with their participation in all stages of NK cell differentiation and in acquisition of functional properties by NK cells [28]. Transcription factor IRF-2, apparently, participates in maintenance of differentiated NK cell pool, because a defect of this protein results in an increase of intensity of NK cell apoptosis [28]. It was also demonstrated in vitro that, as the result of IL-12 impact on mature NK cells, an expression of transcription factor ID3 mRNA [7].…”

“…An expression of Gata-3 transcription factor in NK cell progenitors decreases for the short time and then restores to the initial level. High expression of EGR-2, ID-2, T-bet, and Gata-3 factors is connected with their participation in all stages of NK cell differentiation and in acquisition of functional properties by NK cells [28]. Transcription factor IRF-2, apparently, participates in maintenance of differentiated NK cell pool, because a defect of this protein results in an increase of intensity of NK cell apoptosis [28].…”

“…Thus, NK cell differentiation process in humans is connected with activation of transcriptional factors E4BP4, TOX, Ikaros, EGF1, PU.1, EGR-2, ID-2, IRF-2, T-bet, Gata-3 [28], and consequential change of repertoire of cell surface receptors, particularly acquirement of receptors CD56, CD94/NKG2A, NKp46, NKG2D, NKp30, NKp44, receptors KIR, CD16 [14,20,28] …”

“…Using the analysis of expressed mRNA in the in vitro model of human NK cells differentiation obtained from umbilical vein, it was determined that at the stage of transfer from stem cells to NK cell progenitors, E4BP4 and TOX transcription factors participate [28]. While differentiation of NK cell progenitors into mature NK cells an expression of the transcription factors Ikaros, EGF1 and PU.1 is observed [28]. There is a high expression of transcription factors EGR-2, ID-2, and T-bet at all stages of NK cell differentiation.…”

Expression of stem cell markers: OCT4, KIT, ITGA6, and ITGB1 in the male germinal epithelium

“…Human CD34 + were isolated as previously described [Pinho et al 2011]. Briefly, mononuclear cells were isolated from umbilical cord blood by gradient centrifugation and then subjected to a MACS system (Miltenyi Biotec, Auburn, CA, USA), using a CD34 epitope QBEND/10 to immunomagnetically select the Biomarkers expression in human germinal epithelium 239…”