Dissection of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic Stem Cells Via Motif-Engineered Chimeric Receptors

Koichiro Saka,Teruyuki Nagamune,Makoto Otsu,Masahiro Kawahara,Chen-Yi Lai,Masanori Nojima,Hiromitsu Nakauchi

doi:10.1007/s12015-017-9768-7

Koichiro Saka, Teruyuki Nagamune + Show 5 more

Open Access

https://doi.org/10.1007/s12015-017-9768-7

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Abstract

Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

Highlights

Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to differentiate into blood cells of multiple lineages [1,2,3,4]
With the use of a prototype chimeric receptor (CR), single-chain Fv (ScFv)/c-Mpl (S-Mpl), we showed that bovine serum albumin (BSA)-Fluo stimulation in Ba/F3 cells activated the signaling molecules Stat1, Stat3, Stat5, PI3K, and Shc, all known as TPO-related signal proteins [20, 21]
Using our unique CR system [18,19,20] and retroviral-mediated transduction techniques developed in our laboratory [29, 30], we investigated how selective/specific activation of signaling molecules downstream of c-Mpl affected abilities of highly purified HSCs in short-term culture

Summary

Introduction

Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to differentiate into blood cells of multiple lineages [1,2,3,4]. Almost all expansion protocols so far use multiple cytokines, generally including a combination of stem cell factor (SCF) and thrombopoietin (TPO). These two cytokines in combination induce in vitro self-renewal in purified murine HSCs [11]. To understand how signals downstream from these cytokine receptors affect stem cell activity remains critical to better clinical use of HSCs. The receptors of SCF and TPO are cKit and c-Mpl, respectively [12, 13]. Using the known consensus motifs in cytokine receptors, we established a chimeric receptor (CR) system with single-chain Fv (ScFv)/ cytokine receptor chimeras capable of motif-specific recruitment of downstream molecules upon stimulation with the artificial ligand BSA-Fluo, viz., fluorescein (Fluo)-conjugated bovine serum albumin (BSA) [18,19,20]. Stimulation of the S-Mpl CR containing a motif known to recruit STAT5 led to highly specific phosphorylation of Stat5 [20]

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