Methods in Molecular Biology (2022) 2436: 55–66

DOI 10.1007/7651_2021_412

© Springer Science+Business Media, LLC 2021

Published online: 10 August 2021

High-Efficiency Differentiation of Human Pluripotent Stem

Cells to Hematopoietic Stem/Progenitor Cells in Random

Positioning Machine Bioreactors

Xiaohua Lei, Chiyuan Ma, Yujing Cao, Yue Xiong, Jian V. Zhang,

and Enkui Duan

Abstract

Human pluripotent stem cells (PSCs) are known to differentiate into almost all the blood lineage cells

in vitro and hold a great promise for studying human early hematopoietic development and have a huge

potential in the treatment of hematological disorders. Although several methods of hematopoietic stem/

progenitor cell (HSPC) differentiation have been developed, the HSPC yields achieved using these

strategies are not yet available for clinical application. Recently, bioreactor-based devices and biochemical

factors synergistically have been used to induce hematopoietic differentiation and showed a potential role in

hematopoiesis. This chapter describes a protocol for using a random positioning machine bioreactor to

culture human PSCs and the large-scale production of HPCs. Techniques for characterizing the differ-

entiated cells and assessing the efficiency of hematopoietic differentiation in the bioreactor with immunos-

taining and flow cytometry are also presented.

Key words Differentiation, Hematopoietic stem/progenitor cells, Human pluripotent stem cell,

Random positioning machine

1

Introduction

Human pluripotent stem cells (hPSCs) include human embryonic

stem cells (ESCs) and induced pluripotent stem cells (iPSCs),

which have the capacity of self-renewal and can differentiate into

almost all blood lineage cells, thus offering an invaluable model for

dissecting early human hematopoietic development and the in vitro

production of hematopoietic stem/progenitor cells (HSPCs) and

functional blood cells for therapies of various hematologic disor-

ders [1–3]. Some in vitro directed hematopoietic differentiation

protocols from hPSCs have been developed, yet to date it remains a

great challenge to generate HSPCs with robust multilineage

engraftment potential and infusion dosage levels of functional

blood cells from hPSCs [4]. Kaufman et al. reported a strategy for

the first time to generate hematopoietic progenitors derived from

human embryonic stem cells by coculturing with the murine S17

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