Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells In Vitro than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

Martha Luevano,Adrian Thrasher,Sophie Derniame,Sergio Querol,Nicola Jackson,Michael Blundell,Michelle Escobedo-Cousin,Anna Domogala,Alejandro Madrigal,Aurore Saudemont,Isabela Pedroza-Pacheco,Zoran Ivanovic

doi:10.1371/journal.pone.0087086

Martha Luevano, Adrian Thrasher + Show 10 more

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https://doi.org/10.1371/journal.pone.0087086

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Abstract

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34+) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34+) and frozen PBCD34+ to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34+ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34+ cultures. NK cells generated from CBCD34+ and PBCD34+ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34+-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34+-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34+ for the production of NK cells in vitro results in higher cell numbers than PBCD34+, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.

Highlights

Natural Killer (NK) cells can kill infected or transformed cells without prior sensitization, making them an ideal cell product for immunotherapy [1]
We did not find any difference in degranulation, killing of K562 cells or antibody dependent cell cytotoxicity (ADCC) by NK cells generated from fresh or frozen CBCD34+ (Figure 1E–G), suggesting that these phenotypic differences did not impact on the functions of the NK cells
Our study revealed that frozen CBCD34+ contained a higher percentage of CD45+CD7+ cells as compared to fresh CBCD34+ and PBCD34+ cells (Figure S5), suggesting that a higher number of NK cell committed progenitor cells were found in frozen CBCD34+

Summary

Introduction

Natural Killer (NK) cells can kill infected or transformed cells without prior sensitization, making them an ideal cell product for immunotherapy [1]. Several studies have explored the possibility of using NK cells for immunotherapy and highlighted the need to obtain high numbers of NK cells with optimal effector functions [2,3,4]. In this context, different sources of HSC have been used to generate NK cells in vitro including bone marrow (BM) [5,6], human embryonic stem cells (hESC) [7,8], mobilized peripheral blood stem cells (PBCD34+) [9,10] and umbilical cord blood stem cells (CBCD34+) [11,12,13,14].

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