(E-175) Enhanced presentation of notch1 and α4β1 integrin ligands by interfacial polymeric coatings improves the emergence of T cell progenitors from human hematopoietic and pluripotent stem cells

Introduction: Therapeutic T-cell engineering in vitro from human primary hematopoietic stem cells (HSCs) or human pluripotent stem cells (PSCs) has relied on recapitulating critical molecular features of the thymus using either feeder stromal cell-based systems that ectopically express notch1 ligand delta-like ligand-4 (DLL-4) or surface immobilized DLL-4 and vascular adhesion protein-1 (VCAM-1). Here, we describe a reductionist engineered thymic niche (ETN) by surface modification of cell culture substrates with an interfacial polymeric coating (IPC), followed by surface immobilization of DLL-4 and VCAM-1. In combination with three-stage dosing of defined lymphopoietic cytokine milieus, IPC-enhanced presentation of DLL-4 and VCAM-1 in our ETN significantly improves the differentiation velocity and yield of T-cell progenitors from primary CD34+ HSCs and PSCs. Our ETN generates CD4+/CD8+CD3+aßTCR+ T-cells at a greater magnitude than conventional ETNs, providing a facile means to engineer T-cells.

Materials and Methods: IPC-ETN preparation: Poly-L-ornithine (PLO) was dissolved in sterile deionized (DI) water at a concentration of 100 ug/mL, and tissue culture plates were coated with PLO solution overnight at 4C. After overnight incubation, the PLO solution was aspirated, the plates washed with sterile DI water twice, followed by surface immobilization of DLL-4 and VCAM-1 dissolved in sterile PBS at a solution concentration of 12.5 ug/mL and 2.5 ug/mL, respectively for additional overnight incubation at 4C. The protein solution was aspirated and the plates were washed once with sterile PBS prior to CD34+ HSC seeding. Cell culture: CD34+ HSCs isolated from cord blood or derived from PSCs were seeded in IPC-ETNs at a density of 3e5 cells/mL in a T-cell differentiation media comprised of Penn/strep, BIT 9500, b-ME, L-AA, SCF, Flt3L, IL-3, IL-7, TNF-a, and CXCL12 at staged, varying concentrations over the course of 14-32 days. Media was exchanged once a week. Cells were characterized with flow cytometry.

Results, Conclusions, and Discussions: We improve upon current ETNs by modifying cell culture substrates with IPCs to enhance the presentation of DLL-4 and VCAM-1. We observe that IPC presentation of DLL-4 and VCAM-1 in our ETN significantly perturbs T-cell differentiation velocity and trajectory from CD34+ HSCs and PSCs into CD4+ and CD8+ T-cells by increasing the strength and avidity of notch1-DLL-4 and α4β1-VCAM-1 interactions. We find that our IPC-ETN robustly generates CD4+ and CD8+ single positive T-cells from diverse primary HSCs and PSC sources at unprecedented rates. Incorporating IPC-ETN into a 2-dimensional endothelium-to-hematopoietic transition (EHT) in PSC-derived hemogenic endothelial cells leads to the significant generation of T–competent cells from PSCs. Surface analysis of our IPC-ETN suggests that the IPC not only improves ligand immobilization through energy minimization but also strengthens the cooperative binding affinity of notch1 and α4β1-integrin receptors to DLL-4 and VCAM-1, respectively.

Acknowledgements (Optional): We thank Dr. Bryan A. Nerger (Harvard University) for insight and discussion on contact angle measurements, and Dr. Kevin E. Healy (University of California, Berkeley) for his valuable insight on analysis for Langmuir protein adsorption isotherms and QCM-D. We thank Azeem Sharda, Dr. Karin Gustafsson, and Dr. David T. Scadden of Harvard Medical School for contributing protocols and intellectual expertise on primary HSC isolation, cell culture, and T-cell development. We thank Dr. John T. Ngo (Boston University), Dr. Tetsuhiro Harimoto (Wyss Institute), Dr. Yoav Binenbaum (Wyss Institute), and Dr. Kwasi Adu-Berchie (Wyss Institute) for scientific discussions. We thank Maurice Perez, Eric Zigon, and Michael Carr of the Wyss Institute for technical staff support. We thank the Harvard John A. Paulson School of Engineering and Applied Sciences Active Learning Laboratory and Makerspace for technical staff support. We thank the Center for Macromolecular Interactions at Harvard Medical School for technical staff support. We thank the Dana-Farber Cancer Institute’s Pasquarello Tissue Bank for providing cord blood, peripheral blood, and bone marrow samples for HSC isolation. We especially hold great appreciation for the patients who donated these aforementioned samples. We acknowledge funding from the NSF (MRSEC), Wellcome Leap Fund (HOPE), and NIH/NCI (i3 center).

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