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3D iPSC-differentiation model
HSC are only generated at later stages by the definitive hematopoietic program.2 In several studies, the hemogenic endothelium (HE), which is predominantly present in the AGM, has been identified as the primary cell source responsible for the emergence of definitive hematopoietic cells.3-5 The emergence of hematopoietic cells from the HE (also referred to as endothelial to hematopoietic transition [EHT]), is controlled by distinct cell-intrinsic signals medi- ated by transcription factor (TF) activation, as well as important cell-extrinsic factors such as cell-cell interac- tions, extracellular matrix molecules, and cytokines.6
The latter are of great interest as diverse cytokines have been shown to regulate the differentiation of mature blood cells from adult HSC.7 Among others, interleukin-3 (IL-3), formerly known as multi colony stimulating factor, has been implicated in survival, proliferation, and differen- tiation of HSC and hematopoietic progenitor populations.8 Although disruption of IL-3 or IL3ra/b genes is not lethal in mice,9,10 IL-3 has been shown to induce amplification of the HSC pool in the midgestation mouse embryo.11 The same study suggests that the mechanism by which IL-3 expression facilitates the detection of HSC in early embryonic day (E) E10 AGM and yolk sac (YS) is mediated by supporting (i) either the survival and/or pro- liferation of a limiting number of previously undetectable HSC or (ii) even the emergence of new HSC. Along the same lines, IL-3 has been shown to positively regulate hemangioblast development in the murine AGM, by stim- ulating the differentiation and proliferation of fetal liver kinase (Flk) 1+ mesodermal progenitors.12
While most of these findings are derived from non-ver- tebrate or murine studies, little is known about the differ- ent cell-intrinsic and extrinsic regulators of human embry- onic hematopoietic development. Similarly, the role of IL-3 in human hematopoietic specification has remained elusive. Given the difficulties to study these processes in humans, in vitro hematopoietic differentiation of human pluripotent stem cells (hPSC) provides an invaluable plat- form to study hematopoietic specification and identify factors critically regulating the human EHT.13,14 In vitro hPSC-derived hematopoietic development has been shown to proceed through distinct stages including KDR+ mesodermal progenitors, hemogenic endothelium and finally the emergence of hematopoietic stem/progenitor cells. Whereas the two waves of primitive and definitive hematopoietic development are spatiotemporally regulat- ed in vivo,15 hPSC-derived hematopoiesis often yields a mixture of cells derived from the primitive and definitive hematopoietic program.6,16 Several modulators, such as the Activin/Nodal- and WNT-signaling pathways, have been identified to be important for the emergence of definitive hematopoietic precursor cells.17 However, even when con- sidering the most advanced in vitro differentiation proto- cols available to date, only very limited hematopoietic engraftment and a restriction to the myeloid differentia- tion fate have been observed for hPSC-derived HSC,6 highlighting the necessity to better characterize PSC- derived hematopoiesis.
One factor often neglected in PSC-based hematopoietic differentiation systems is the influence of the microenvi- ronment. The development of HSC in the AGM is critical- ly regulated by microenvironmental signals from the sur- rounding mesenchyme, hematopoietic and endothelial cells.18 In order to gain further insights into the cues regu- lating early developmental processes, different organoid
systems have been introduced and shown to closely mimic features of e.g., lung, intestine or brain develop- ment.19 Organoid-based hematopoietic differentiation sys- tems providing a 3-dimensional (3D) niche for developing HSC may also be of high relevance to gain novel insights into transcriptional regulators of human hematopoietic development.
Considering the important role of IL-3 in early murine hematopoietic development, we sought to evaluate the regulatory role of IL-3 in early human hematopoiesis. Using an organoid-like hematopoietic differentiation pro- tocol, we demonstrate that the addition of IL-3 alone is sufficient to induce the continuous production of imma- ture, myeloid progenitor cells. We furthermore provide evidence that EHT is IL-3 dependent, highlighting a very early effect of this cytokine on hematopoietic specifica- tion of hPSC. Thus, we have developed an organoid-like hematopoietic differentiation system for hPSC and unrav- el the IL-3 signaling cascade as an important regulator of early human hematopoietic specification.
Methods
Human induced pluripotent stem cell cultivation
hPSC were cultured on irradiated murine embryonic fibroblasts (MEF) derived from CF1 mice in induced PSC (iPSC)-medium (knockout DMEM, 20% knockout serum replacement, 1 mM L- glutamine, 1% NEAA, 1% penicillin/streptomycin [all Invitrogen], 0.1 mM b-mercaptoethanol [Sigma-Aldrich], and 10 ng/mL basic fibroblast growth factor [bFGF] [Peprotech]).20 Cells were passaged every 7-10 days using incubation with collagenase IV.
Human induced pluripotent stem cell differentiation
Hematopoietic differentiation was performed following our establish protocol.21 In brief, embryoid bodies (EB) were generated from iPSC colonies disrupted with collagenase by cultivation of the fragments on an orbital shaker (85 rpm) in iPSC-medium with- out bFGF but supplemented with 10 mM Rock inhibitor (Y-27632; Tocris). Medium was changed after 2-3 days. After 5 days, EB were manually selected and transferred to adherent plates in dif- ferentiation medium I (APEL [II] medium, Stem Cell Technologies or prepared according to a previously published protocol22) supple- mented with 50 ng/mL IL-3, a combination of 25 ng/mL)22 IL-3 and 50 ng/mL M-CSF, 250 ng/mL of a rat anti-human IL-3 anti- body (Clone BVD8-3G11, BD Bioscience) or without cytokine addition. Alternatively, the last step of differentiation was carried out in suspension as described recently.23 Floating hematopoietic cells were harvested from the medium and filtered through a 150- μm mesh from day 14 onwards while hemanoids remained in cul- ture.
For terminal differentiation, cells harvested from the hemanoids were cultured in differentiation medium II (RPMI 1640 medium supplemented with 10% fetal serum, 2 mM L-glutamine, 1% penicillin-streptomycin, all from Invitrogen) supplemented with 100 ng/mL human macrophage (hM) colony stimulating factor (CSF) (hM-CSF), human granulocyte-CSF (hG-CSF), or human macrophage-granuclocyte-CSF (hGM-CSF), for 7–10 days.
Whole transcriptome analysis
Gene expression analyses were performed using Illumina HumanHT12v4 BeadChips® at the Genomics and Proteomics Core facility of the DKFZ-Heidelberg. Gene expression data were quantile-normalized and subsequently anlayzed using R/Bioconductor through the graphical user interface Chipster
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