Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34&lt;sup&gt;+&lt;/sup&gt; Cells

Perdomo, José; Feng, Yuping; Leung, Halina; Chong, Beng H.

doi:10.3791/56420

Cited by 18 publications

(26 citation statements)

References 22 publications

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“…Differentiation of CD34 + HSPCs into platelets in vitro. Platelet differentiation protocol was adapted from Perdomo et al 48 . Briefly, day 4 post-editing WAS CD34 + HSPCs were seeded at a density of 1 × 10 6 cells per ml in StemSpan ACF medium supplemented with TPO (50 ng/ml) at 37°C/5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome

et al. 2020

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Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.

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Section: Methodsmentioning

confidence: 99%

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome

et al. 2020

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“…Human MKs were differentiated from umbilical CB-derived CD34 þ HSCs according to previously published methods. 35,36 Umbilical CB samples from full-term pregnancies were obtained from the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. Ficoll-Paque (GE Healthcare, Marlborough, Massachusetts, United States) was used to purify the mononuclear cells from the CB samples.…”

Section: Cd34 þ Hsc Differentiation Into Mks In Vitromentioning

confidence: 99%

“…MK ploidy was determined according to previously described methods with minor modifications. 35,36 MKs were harvested and labeled with an APC-conjugated integrin αIIb antibody for 30 minutes at 4°C. The cells were then washed twice with PBS containing 0.5% BSA and 2 mM EDTA and resuspended in hypotonic citrate buffer (1.25 mM sodium citrate, 2.5 mM sodium chloride, 3.5 mM dextrose) containing 20 μg/mL propidium iodide (PI) solution, 10 U/mL RNase A, and 0.05% TritonX-100.…”

Section: Flow Cytometry To Investigate Mk Ploidy Cell Apoptosis Andmentioning

confidence: 99%

Ibrutinib Suppresses Early Megakaryopoiesis but Enhances Proplatelet Formation

Huang

et al. 2020

Thromb Haemost

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Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase, has a favorable safety profile in patients with B cell-related malignancies. A primary adverse effect of ibrutinib is thrombocytopenia in the early stages of treatment, but platelet counts increase or recover as treatment continues. Currently, the effects of ibrutinib on megakaryopoiesis remain unclear. In this study, we investigated the mechanism by which ibrutinib induces thrombocytopenia using cord blood CD34+ hematopoietic stem cells (HSCs), a human megakaryoblastic cell line (SET-2), and C57BL/6 mice. We show that treatment with ibrutinib can suppress CD34+ HSC differentiation into megakaryocytes (MKs) and decrease the number of colony-forming unit-MKs (CFU-MKs). The ibrutinib-dependent inhibition of early megakaryopoiesis seems to mainly involve impaired proliferation of progenitor cells without induction of apoptosis. The effects of ibrutinib on late-stage megakaryopoiesis, in contrast to early-stage megakaryopoiesis, include enhanced MK differentiation, ploidy, and proplatelet formation in CD34+ HSC-derived MKs and SET-2 cells. We also demonstrated that MK adhesion and spreading, but not migration, were inhibited by ibrutinib. Furthermore, we revealed that integrin αIIbβ3 outside-in signaling in MKs was inhibited by ibrutinib. Consistent with previous clinical observations, in C57BL/6 mice treated with ibrutinib, platelet counts decreased by days 2 to 7 and recovered to normal levels by day 15. Together, these results reveal the pathogenesis of ibrutinib-induced transient thrombocytopenia. In conclusion, ibrutinib suppresses early megakaryopoiesis, as evidenced by inhibition of MK progenitor cell proliferation and CFU-MK formation. Ibrutinib enhances MK differentiation, ploidy, and proplatelet formation, while it impairs integrin αIIbβ3 outside-in signaling.

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“…In the literature, different cytokine combinations have been reported that promote differentiation towards either megakaryocytic, erythroid, or granulo-monocyte lineages [19][20][21][22] . An advantage of this protocol is that it allows differentiation along all four myeloid populations, namely monocytes, granulocytes, megakaryocytes, and erythrocytes.…”

Section: Discussionmentioning

confidence: 99%

Pan-myeloid Differentiation of Human Cord Blood Derived CD34<sup>+</sup> Hematopoietic Stem and Progenitor Cells

Bapat

Keita

Sharma

2019

JoVE

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Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34 + hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34 + cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34 + cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

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Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34<sup>+</sup> Cells

Cited by 18 publications

References 22 publications

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome

Ibrutinib Suppresses Early Megakaryopoiesis but Enhances Proplatelet Formation

Pan-myeloid Differentiation of Human Cord Blood Derived CD34<sup>+</sup> Hematopoietic Stem and Progenitor Cells

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