Induced Pluripotent Stem Cell-Derived Neutrophil Granulocytes - Flow Cytometry Analysis Reveals Distinct Subpopulations

Severe congenital neutropenia (SCN) comprises a spectrum of monogenic disorders characterized by impaired differentiation and function of neutrophil granulocytes. Since animal models often do not fully recapitulate human SCN phenotypes and primary bone marrow samples of patients are scarce, alternative strategies are desirable to study the genetic causes and mechanisms. Induced pluripotent stem cells (iPSCs) can be differentiated into neutrophil granulocytes, thereby presenting an excellent tool to study hematopoiesis and especially neutrophil differentiation in health and disease in vitro.

Recently, neutrophil progenitors and mature neutrophils of murine and human bone marrow have been characterized by single-cell RNA sequencing, mass cytometry and flow cytometry and are now referred to as proNeu, preNeu, immature-Neu and mature-Neu, at least in part reflecting conventional morphological classification of myeloblasts, promyelocytes, myelocytes/metamyelocytes and band and segmented neutrophils, respectively.

Here we developed a flow cytometry antibody panel and gating strategy, which robustly identified distinct myeloid subsets in iPS-derived neutrophils. We adopted a differentiation protocol, which consists of feeder- and serum-free differentiation of iPS cells by mesoderm induction and patterning, followed by lineage progression through hemogenic endothelium to hematopoietic progenitors and finally mature neutrophil granulocytes. Floating cells arise, which can be harvested continuously and analyzed by flow cytometry.

Based on expression of cell surface molecules, we defined four subpopulations: After selecting for single, live, CD45 ⁺ and CD14 ^- cells, the different progenitor stages were first defined by their expression of CD117 and CD49d. CD117 ^midCD49d ^high cells were further stratified into SSC ^lowCD34 ⁺ cells and SSC ^highCD34 ^- cells, representing myeloblasts (proNeu1) and promyelocytes (proNeu2/preNeu), respectively. These cells progressed to CD117 ^-CD49d ^mid and were CD11b ⁺CD101 ⁺, which defines myelocytes/metamyelocytes (immature-Neus). CD117 ^-CD49d ^low cells were CD11b ⁺CD101 ⁺ and expressed additionally CD16, resembling band/segmented neutrophils (mature-Neus). Additionally, these iPS-derived cells progressively expressed CD35, which is also a maturation marker of human myeloid cells in vivo. May-Grünwald-Giemsa staining of these four subpopulations (CD117 ^midCD49d ^highSSC ^lowCD34 ⁺, CD117 ^mid CD49d ^highSSC ^highCD34 ^-, CD117 ^- CD49d ^midand CD117 ^- CD49d ^low) revealed homogenous populations of sorted cells, morphologically resembling myeloblasts, promyelocytes, myelocytes/metamyelocytes and band/segmented neutrophils, respectively.

Ongoing studies in our lab make use of this model to a) validate the functional significance of rare genetic variants and b) further assess transcriptomic and proteomic changes on a single cell level. Thus, we provide a promising tool to study neutrophil differentiation in health and disease.

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