Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines

Mills, Jason A.; Wang, Kai; Paluru, Prasuna; Lei, Ying; Lin, Li; Galvão, Aline M.; Xu, Dongpo; Yao, Yu; Sullivan, Spencer K.; Sullivan, Lisa; Mac, Helen; Omari, Amel; Jean, Jyh Chang; Shen, Steven S.; Gower, Adam C.; Spira, Avi; Mostoslavsky, Gustavo; Kotton, Darrell N.; French, Deborah L.; Weiss, Mitchell J.; Gadue, Paul

doi:10.1182/blood-2013-02-484444

Cited by 76 publications

(65 citation statements)

References 18 publications

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“…During the course of this study, serial Western blotting for GATA1 showed that female GATA1s iPSC lines did not undergo reactivation of the lyonized X chromosome, which can occur during iPSC passage (19,22). Genetic and epigenetic variation between different iPSC lines may alter their developmental potential, including hematopoietic (23,24). We controlled for this by systematically analyzing multiple iPSC clones from different individuals of the same relevant genotype ( Figure 1, C and D, n = 6 GATA1s and 12 WT GATA1) and isogenic lines from TMD blasts (T21/GATA1s) and normal blood cells (T21/WT GATA1) of the same DS patients (n = 2 different subjects, Supplemental Table 1 and Figure 1, G and H).…”

Section: Generation Of Ipscsmentioning

confidence: 81%

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

Byrska-Bishop¹,

VanDorn²,

Campbell³

et al. 2015

J. Clin. Invest.

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Section: Generation Of Ipscsmentioning

confidence: 81%

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

Byrska-Bishop¹,

VanDorn²,

Campbell³

et al. 2015

J. Clin. Invest.

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“…23 iPSCs were differentiated into megakaryocytes as previously described. 24 For all, large megakaryocytes were isolated using a 2-step bovine serum albumin density gradient described for murine megakaryocytes 19 and counted by hematocytometer before retro-orbital infusion in 200 mL phosphate-buffered saline (PBS; Invitrogen). Similar AMC growth conditions were used to isolate EV-PLPs.…”

Section: Methodsmentioning

confidence: 99%

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

Wang

Hayes

Jarocha

et al. 2015

Blood

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• Infused human megakaryocytes release young platelets in the lungs with characteristics similar to donor platelets.• Platelets released from ex vivo-derived megakaryocytes are preactivated and compare poorly to donor platelets.Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. (Blood. 2015;125(23):3627-3636)

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“…12 Hemizygous insertions were confirmed by using Southern blot analysis 12,13 (supplemental Figure 2B-C). iPSCs were differentiated into hematopoietic progenitor cells (HPCs) and megakaryocytes by using a previously described protocol 14 and were analyzed for both quantitative gene expression and surface marker expression. The functionality of agonist-stimulated megakaryocytes was evaluated by flow cytometry using PAC-1 antibody, 15 which binds specifically to the active conformation of integrin aIIbb3 and The publication costs of this article were defrayed in part by page charge payment.…”

Section: Methodsmentioning

confidence: 99%

High-level transgene expression in induced pluripotent stem cell–derived megakaryocytes: correction of Glanzmann thrombasthenia

Sullivan¹,

Mills

Koukouritaki

et al. 2014

Blood

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• When targeted to a single allele of the AAVS1 locus, the Gp1ba promoter drives a high level of expression specifically to megakaryocytes.• Transgene rescue in iPSCs provides a model for the return of surface aIIbb3 expression to near-normal levels in patients with type I GT.Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin aIIbb3. Hemizygous insertion of Gp1ba promoter-driven human aIIb complementary DNA into the AAVS1 locus of iPSCs led to high aIIb messenger RNA and protein expression and correction of surface aIIbb3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin aIIbb3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function. (Blood. 2014;123(5):753-757)

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Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines

Abstract: Key Points Normal induced pluripotent stem cells exhibit donor-specific gene expression signatures and the capacity for hematopoietic development. CNVs acquired during reprogramming or selection of rare CNVs present in the starting cell population may alter iPSC developmental potential.

Cited by 76 publications

References 18 publications

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

High-level transgene expression in induced pluripotent stem cell–derived megakaryocytes: correction of Glanzmann thrombasthenia

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