Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

Abstract

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.

Figure 1

Schematic diagram of the protocol used to obtain multipotent lin⁻CD34⁺CD43⁺CD45⁺ progenitors from human pluripotent stem cells. (a) Undifferentiated hiPSCs (iPS(Foreskin)-1) cocultured with (b) overgrown OP9. (c) After 8 days of coculture differentiated blood-forming hPSC colonies with radial sac-like structures are formed. (d) and (e) Typical flow cytometric analysis of differentiated iPS(Foreskin)-1 cells collected after 8 days of OP9 cocluture shows that hiPSC-derived CD34⁺ cells consist of CD31⁺CD43⁻ endothelial cells, CD43⁺ hematopoietic cells, and CD31⁻CD43⁻ mesenchymal cells. Three major subsets of hematopoietic progenitors could be defined within CD43⁺ population: CD235a/41a⁺ erythro-megakaryocytic progenitors, and CD235a/CD41a⁻CD45⁻ and CD235a/CD41a⁻CD45⁺ multipotent progenitors. For the analysis, live (7-AAD⁻) and human (TRA-1-85⁺) cells are gated. (f) When differentiated hiPSCs are collected on day 8 of OP9 coculture by enzymatic digestion and cultured in nonadherent conditions in the presence of GM-CSF, cells spontaneously form large aggregates with hematopoietic cells released from these aggregates into suspension. (g) Typical flow cytometric analysis of cells cultured with GM-CSF for 2 days shows a marked expansion of the CD45⁺ population of hematopoietic progenitors within the CD43⁺ population. (h) After Percoll separation and (i) magnetic sorting a population of CD45⁺ progenitors with greater than 90% purity can be obtained. (j) Isolated CD45⁺ progenitors do not express major lineage markers, but retain expression of CD34, CD90, and CD177 markers characteristic of primitive hematopoietic cells. Scale bars=300µm.

Figure 2

Directed differentiation of lin⁻CD34⁺CD43⁺CD45⁺ progenitors into mature myelomonocytic cells. Left-hand side: hPSC-derived lin⁻CD34⁺CD43⁺CD45⁺ progenitors are shown as cytospin after magnetic sorting (scale bar=30µm). Right-hand images: Cultures (scale bars=300µm), cytospins (scale bars=10µm) and flow cytometric analyses show the morphologic and phenotypic characteristics of mature myelomonocytic cells. * represents intracellular staining for flow cytometry. Bellow dashed line: Osteoclast precursors were expanded without RANKL for 4–5 days (scale bars=300µm). Multi-nucleated mature osteoclasts are TRAP⁺ in maturation condition. Scale bars=300µm.

Figure 3

Timing for directed differentiation of mature myelomonocytic cells from human pluripotent stem cells. To induce hematopoietic differentiation, hES/iPSCs are collected from MEF cocultures and transferred onto overgrown OP9 cells. After 9 days of coculture with OP9, cells should be collected and treated with enzymes to prepare single cell suspensions containing lin⁻CD34⁺CD43⁺CD45⁺ hematopoietic progenitors. To expand these progenitors, single cells should be cultured in non-adherent conditions (pHEMA-coated flasks) in the presence of GM-CSF for 2 days. Expanded lin⁻CD34⁺CD43⁺CD45⁺ progenitors enriched in myeloid CFCs should be isolated by magnetic sorting. Through cell culture with a particular cytokine combination, myeloid progenitors can be differentiated into mature myeloid cells of the desired type. Depending on the cell type, 5 to 19 days of culture are required to obtain a mature cell population.

Figure 4

Demonstration of morphological changes in OP9 associated with a loss of hematopoiesis-inductive properties. (a) Spontaneous adipogenesis in overgrown OP9 cells. (b) After 8 days of hPSC/adipogenic OP9 coculture, the majority of hPSC colonies retain undifferentiated morphology. (c) Heterogeneous overgrown OP9 cultures with spindle cells forming long cords. (d) After 8 days of hPSC/heterogeneous OP9 coculture, the majority of hPSC colonies retain undifferentiated morphology. Scale bars=300µm.