Expansion on Stromal Cells Preserves the Undifferentiated State of Human Hematopoietic Stem Cells Despite Compromised Reconstitution Ability

Mattias Magnusson,Xinmin Li,Maria I Sierra,Ben Van Handel,Rajkumar Sasidharan,Melissa Romero,Hanna K A Mikkola,Sacha L Prashad,Pamela Saarikoski,Andy Huang,Zoran Ivanovic

doi:10.1371/journal.pone.0053912

Abstract

Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38−CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38−CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

Highlights

Hematopoietic stem cells (HSC) have been successfully used to treat leukemias, inherited immune deficiencies and other lifethreatening blood diseases [1,2]
To define the extent to which HSC properties can be maintained in culture, we first assessed the ability of different stromal cell lines to preserve the undifferentiated state of human HSC (Figure 1A, Figure S1 and data not shown)
We found that the supportive OP9M2 stroma has mesenchymal stem cell (MSC) characteristics and can generate osteoblasts, adipocytes and chondrocytes (Figure S3), as reported for the OP9 bulk cells [32], which is intriguing since MSCs and their derivatives are key components of the bone marrow HSC niche in vivo [6,8,9]

Summary

Introduction

Hematopoietic stem cells (HSC) have been successfully used to treat leukemias, inherited immune deficiencies and other lifethreatening blood diseases [1,2]. Only a fraction of patients benefit from this therapy due to the lack of HLA-matched bone marrow donors, and low number of HSC in cord blood [3]. There has been little success in expanding human HSC for clinical purposes due to limited understanding of the complex mechanisms governing HSC properties, and how these programs become compromised in culture. Most HSC regulators have been identified using gene-targeted mouse models [4], whereas mechanistic understanding of human hematopoiesis is lagging behind due to lack of suitable in vitro and in vivo model systems for manipulating human HSC or their niche. A major challenge in culturing HSC is the difficulty to recreate the specialized microenvironment that regulates self-renewal of HSC within hematopoietic tissues; as a result, cultured HSC are subjected to rapid differentiation or death [5]

Methods

Results

Conclusion