mRNA-mediated glycoengineering ameliorates deficient homing of human stem cell–derived hematopoietic progenitors
Jungmin Lee, … , Robert Sackstein, Derrick J. Rossi
Jungmin Lee, … , Robert Sackstein, Derrick J. Rossi
Published May 8, 2017
Citation Information: J Clin Invest. 2017;127(6):2433-2437. https://doi.org/10.1172/JCI92030.
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Brief Report

mRNA-mediated glycoengineering ameliorates deficient homing of human stem cell–derived hematopoietic progenitors

Abstract

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell–derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin–bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.

Authors

Jungmin Lee, Brad Dykstra, Joel A. Spencer, Laurie L. Kenney, Dale L. Greiner, Leonard D. Shultz, Michael A. Brehm, Charles P. Lin, Robert Sackstein, Derrick J. Rossi

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Figure 3

FUT6 modified mRNA enhances BM homing and extravasation of xenotransplanted hiPS-HSPCs.

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FUT6 modified mRNA enhances BM homing and extravasation of xenotransplan...
(A) Dye-labeled FUT6- (DiD, blue) or control modified mRNA–transfected (DiI, green) hiPS-HSPCs mixed at a 1:1 ratio and imaged before transplant. (B) 3D reconstruction of a calvarium section of an NSG mouse transplanted with DiD- and DiI-labeled hiPS-HSPCs. Image represents view of sagittal plane, with a portion of bone digitally removed to optimize visualization. Imaging depth extends to approximately 150 μm beneath the bone. (C) FUT6 modified mRNA–transfected hiPS-HSPCs showed increased homing at 2 hours after transplant compared with control modified mRNA–transfected cells. n = 3 independent experiments (6 mice). (D) Image taken 24 hours after transplant showing DiD- (blue) and DiI-labeled (green) hiPS-HSPCs and AngioSense-labeled blood vessels (red). Extravasated hiPS-HSPCs were defined as those completely distinct from the blood vessel. White arrows indicate non-extravasated cells; yellow arrows indicate extravasated cells. (E) FUT6 modified mRNA transfection increases the percentage of homed hiPS-HSPCs that extravasated from the calvarial blood vessels. n = 3 independent experiments (5 mice). (F) Representative FACS plots showing human chimerism at 2 weeks after transplant. (G) Time course of peripheral blood chimerism in NSG mice receiving transplant of human CD34+ cord blood (CB) cells, and control or FUT6 modified mRNA–transfected hiPS-HPCs. Scale bars: 50 μm. Error bars in C and E indicate SEM. P values in C and E were calculated by Student’s t test.