Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents

Yanfeng Liu, Ying Wang, Ying Wang, Ying Wang, Yongxing Gao, Yongxing Gao, Jessica A. Forbes, Rehan Qayyum, Lewis Becker, Linzhao Cheng, Linzhao Cheng, Zack Z. Wang, Zack Z. Wang

Research output: Contribution to journalArticle

Abstract

Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animalderived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administrationapproved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin).Weused this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41+CD42a+ MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41+CD42a+ MKs that also expressed theMKmarkers CD42b and CD61 and displayed polyploidy (‡16% of derived cells withDNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo.

Original languageEnglish (US)
Pages (from-to)309-319
Number of pages11
JournalStem cells translational medicine
Volume4
Issue number4
DOIs
StatePublished - 2015

Fingerprint

Induced Pluripotent Stem Cells
Megakaryocytes
United States Food and Drug Administration
Pharmacology
oprelvekin
Blood Platelets
Hematopoietic Stem Cells
Megakaryocyte Progenitor Cells
Interleukin-11
RNA Sequence Analysis
Thrombopoietin
Polyploidy
Gene Expression Profiling
Hemostasis
Platelet Count
Wound Healing
Blood Vessels
Albumins
Cell Culture Techniques
Bone Marrow

Keywords

  • Human induced pluripotent stem cells
  • Megakaryocytes
  • Plasbumin
  • Romiplostim
  • Thrombopoietin

ASJC Scopus subject areas

  • Cell Biology
  • Developmental Biology

Cite this

Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents. / Liu, Yanfeng; Wang, Ying; Wang, Ying; Wang, Ying; Gao, Yongxing; Gao, Yongxing; Forbes, Jessica A.; Qayyum, Rehan; Becker, Lewis; Cheng, Linzhao; Cheng, Linzhao; Wang, Zack Z.; Wang, Zack Z.

In: Stem cells translational medicine, Vol. 4, No. 4, 2015, p. 309-319.

Research output: Contribution to journalArticle

Liu, Yanfeng ; Wang, Ying ; Wang, Ying ; Wang, Ying ; Gao, Yongxing ; Gao, Yongxing ; Forbes, Jessica A. ; Qayyum, Rehan ; Becker, Lewis ; Cheng, Linzhao ; Cheng, Linzhao ; Wang, Zack Z. ; Wang, Zack Z. / Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents. In: Stem cells translational medicine. 2015 ; Vol. 4, No. 4. pp. 309-319.
@article{1e3dee741ca84b7ea08c385f2cd2340d,
title = "Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents",
abstract = "Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animalderived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administrationapproved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin).Weused this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41+CD42a+ MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41+CD42a+ MKs that also expressed theMKmarkers CD42b and CD61 and displayed polyploidy (‡16{\%} of derived cells withDNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo.",
keywords = "Human induced pluripotent stem cells, Megakaryocytes, Plasbumin, Romiplostim, Thrombopoietin",
author = "Yanfeng Liu and Ying Wang and Ying Wang and Ying Wang and Yongxing Gao and Yongxing Gao and Forbes, {Jessica A.} and Rehan Qayyum and Lewis Becker and Linzhao Cheng and Linzhao Cheng and Wang, {Zack Z.} and Wang, {Zack Z.}",
year = "2015",
doi = "10.5966/sctm.2014-0183",
language = "English (US)",
volume = "4",
pages = "309--319",
journal = "Stem cells translational medicine",
issn = "2157-6564",
publisher = "AlphaMed Press",
number = "4",

}

TY - JOUR

T1 - Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents

AU - Liu, Yanfeng

AU - Wang, Ying

AU - Wang, Ying

AU - Wang, Ying

AU - Gao, Yongxing

AU - Gao, Yongxing

AU - Forbes, Jessica A.

AU - Qayyum, Rehan

AU - Becker, Lewis

AU - Cheng, Linzhao

AU - Cheng, Linzhao

AU - Wang, Zack Z.

AU - Wang, Zack Z.

PY - 2015

Y1 - 2015

N2 - Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animalderived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administrationapproved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin).Weused this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41+CD42a+ MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41+CD42a+ MKs that also expressed theMKmarkers CD42b and CD61 and displayed polyploidy (‡16% of derived cells withDNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo.

AB - Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animalderived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administrationapproved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin).Weused this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41+CD42a+ MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41+CD42a+ MKs that also expressed theMKmarkers CD42b and CD61 and displayed polyploidy (‡16% of derived cells withDNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo.

KW - Human induced pluripotent stem cells

KW - Megakaryocytes

KW - Plasbumin

KW - Romiplostim

KW - Thrombopoietin

UR - http://www.scopus.com/inward/record.url?scp=84930478276&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84930478276&partnerID=8YFLogxK

U2 - 10.5966/sctm.2014-0183

DO - 10.5966/sctm.2014-0183

M3 - Article

C2 - 25713465

AN - SCOPUS:84930478276

VL - 4

SP - 309

EP - 319

JO - Stem cells translational medicine

JF - Stem cells translational medicine

SN - 2157-6564

IS - 4

ER -