Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1

Abstract

The molecular and cellular signals that guide T-cell development from hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. The thymic microenvironment integrates multiple niche molecules to potentiate T-cell development in vivo. Recapitulating these signals in vitro in a stromal cell-free system has been challenging and limits T-cell generation technologies. Here, we describe a fully defined engineered in vitro niche capable of guiding T-lineage development from HSPCs. Synergistic interactions between Notch ligand Delta-like 4 and vascular cell adhesion molecule 1 (VCAM-1) were leveraged to enhance Notch signaling and progenitor T-cell differentiation rates. The engineered thymus-like niche enables in vitro production of mouse Sca-1+cKit+ and human CD34+ HSPC-derived CD7+ progenitor T-cells capable of in vivo thymus colonization and maturation into cytokine-producing CD3+ T-cells. This engineered thymic-like niche provides a platform for in vitro analysis of human T-cell development as well as clinical-scale cell production for future development of immunotherapeutic applications.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Optimization of T-cell differentiation in defined medium.
Figure 2: VCAM-1 enhances committed DN3 proT-cell yield in the engineered thymic-like niche.
Figure 3: Human HSPCs generate proT-cells in the engineered thymic-like niche.
Figure 4: Human proT-cells home and mature in vivo.

References

  1. 1

    Komanduri, K.V. et al. Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing. Blood 110, 4543–4551 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Ruggeri, A. et al. Outcomes, infections, and immune reconstitution after double cord blood transplantation in patients with high-risk hematological diseases. Transpl. Infect. Dis. 13, 456–465 (2011).

    CAS  PubMed  Google Scholar 

  3. 3

    Menon, T. et al. Lymphoid regeneration from gene-corrected SCID-X1 subject-derived iPSCs. Cell Stem Cell 16, 367–372 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Zakrzewski, J.L. et al. Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation. Nat. Med. 12, 1039–1047 (2006).

    CAS  PubMed  Google Scholar 

  5. 5

    Schmitt, T.M. & Zúñiga-Pflücker, J.C. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Awong, G. et al. Characterization in vitro and engraftment potential in vivo of human progenitor T cells generated from hematopoietic stem cells. Blood 114, 972–982 (2009).

    CAS  PubMed  Google Scholar 

  7. 7

    La Motte-Mohs, R.N., Herer, E. & Zúñiga-Pflücker, J.C. Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro. Blood 105, 1431–1439 (2005).

    CAS  PubMed  Google Scholar 

  8. 8

    Smith, M.J. et al. In vitro T-cell generation from adult, embryonic, and induced pluripotent stem cells: many roads to one destination. Stem Cells 33, 3174–3180 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Ikawa, T. et al. An essential developmental checkpoint for production of the T cell lineage. Science 329, 93–96 (2010).

    CAS  PubMed  Google Scholar 

  10. 10

    Reimann, C. et al. Human T-lymphoid progenitors generated in a feeder-cell-free Delta-like-4 culture system promote T-cell reconstitution in NOD/SCID/γc(−/−) mice. Stem Cells 30, 1771–1780 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Fernandez, I., Ooi, T.P. & Roy, K. Generation of functional, antigen-specific CD8+ human T cells from cord blood stem cells using exogenous Notch and tetramer-TCR signaling. Stem Cells 32, 93–104 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Delaney, C. et al. Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat. Med. 16, 232–236 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Taqvi, S., Dixit, L. & Roy, K. Biomaterial-based notch signaling for the differentiation of hematopoietic stem cells into T cells. J. Biomed. Mater. Res. A 79, 689–697 (2006).

    PubMed  Google Scholar 

  14. 14

    Petrie, H.T. & Zúñiga-Pflücker, J.C. Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annu. Rev. Immunol. 25, 649–679 (2007).

    CAS  PubMed  Google Scholar 

  15. 15

    Besseyrias, V. et al. Hierarchy of Notch–Delta interactions promoting T cell lineage commitment and maturation. J. Exp. Med. 204, 331–343 (2007).

    PubMed  PubMed Central  Google Scholar 

  16. 16

    Hozumi, K. et al. Delta-like 4 is indispensable in thymic environment specific for T cell development. J. Exp. Med. 205, 2507–2513 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Calderón, L. & Boehm, T. Synergistic, context-dependent, and hierarchical functions of epithelial components in thymic microenvironments. Cell 149, 159–172 (2012).

    PubMed  Google Scholar 

  18. 18

    Salomon, D.R. et al. Vascular cell adhesion molecule-1 is expressed by cortical thymic epithelial cells and mediates thymocyte adhesion. Implications for the function of alpha4beta1 (VLA4) integrin in T-cell development. Blood 89, 2461–2471 (1997).

    CAS  PubMed  Google Scholar 

  19. 19

    Prockop, S.E. et al. Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex. J. Immunol. 169, 4354–4361 (2002).

    CAS  PubMed  Google Scholar 

  20. 20

    Csaszar, E. et al. Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling. Cell Stem Cell 10, 218–229 (2012).

    CAS  PubMed  Google Scholar 

  21. 21

    Koch, U. et al. Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment. J. Exp. Med. 205, 2515–2523 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Andrawes, M.B. et al. Intrinsic selectivity of Notch 1 for Delta-like 4 over Delta-like 1. J. Biol. Chem. 288, 25477–25489 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Schmitt, T.M., Ciofani, M., Petrie, H.T. & Zúñiga-Pflücker, J.C. Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions. J. Exp. Med. 200, 469–479 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Mohtashami, M. et al. Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes. J. Immunol. 185, 867–876 (2010).

    CAS  PubMed  Google Scholar 

  25. 25

    Milne, C.D., Zhang, Y. & Paige, C.J. Stromal cells attract B-cell progenitors to promote B-cell-B-cell contact and maturation. Scand. J. Immunol. 62, 67–72 (2005).

    CAS  PubMed  Google Scholar 

  26. 26

    Varnum-Finney, B. et al. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J. Cell Sci. 113, 4313–4318 (2000).

    CAS  PubMed  Google Scholar 

  27. 27

    Csaszar, E. et al. Blood stem cell fate regulation by Delta-1-mediated rewiring of IL-6 paracrine signaling. Blood 123, 650–658 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Hong, C., Luckey, M.A. & Park, J.H. Intrathymic IL-7: the where, when, and why of IL-7 signaling during T cell development. Semin. Immunol. 24, 151–158 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Frasca, D. et al. IL-11 synergizes with IL-3 in promoting the recovery of the immune system after irradiation. Int. Immunol. 8, 1651–1657 (1996).

    CAS  PubMed  Google Scholar 

  30. 30

    Jalkanen, S. & Jalkanen, M. Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin. J. Cell Biol. 116, 817–825 (1992).

    CAS  PubMed  Google Scholar 

  31. 31

    Petrie, H.T. Cell migration and the control of post-natal T-cell lymphopoiesis in the thymus. Nat. Rev. Immunol. 3, 859–866 (2003).

    CAS  PubMed  Google Scholar 

  32. 32

    Crisa, L. et al. Cell adhesion and migration are regulated at distinct stages of thymic T cell development: the roles of fibronectin, VLA4, and VLA5. J. Exp. Med. 184, 215–228 (1996).

    CAS  PubMed  Google Scholar 

  33. 33

    Kueh, H.Y. & Rothenberg, E.V. Regulatory gene network circuits underlying T cell development from multipotent progenitors. Wiley Interdiscip. Rev. Syst. Biol. Med. 4, 79–102 (2012).

    CAS  PubMed  Google Scholar 

  34. 34

    Awong, G. et al. Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture. Blood 122, 4210–4219 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Ohishi, K., Varnum-Finney, B. & Bernstein, I.D. Delta-1 enhances marrow and thymus repopulating ability of human CD34(+)CD38(−) cord blood cells. J. Clin. Invest. 110, 1165–1174 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Qasim, W. et al. First clinical application of TALEN engineered universal CAR19 T cells in B–ALL. Blood 126, 2046 (2015).

    Google Scholar 

  37. 37

    Poirot, L. et al. Multiplex genome-edited T-cell manufacturing platform for “off-the-shelf” adoptive T-cell immunotherapies. Cancer Res. 75, 3853–3864 (2015).

    CAS  PubMed  Google Scholar 

  38. 38

    Strowig, T. et al. Transgenic expression of human signal regulatory protein alpha in Rag2−/−gamma(c)−/− mice improves engraftment of human hematopoietic cells in humanized mice. Proc. Natl. Acad. Sci. USA 108, 13218–13223 (2011).

    CAS  PubMed  Google Scholar 

  39. 39

    Fares, I. et al. Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 345, 1509–1512 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Storek, J., Gooley, T., Witherspoon, R.P., Sullivan, K.M. & Storb, R. Infectious morbidity in long-term survivors of allogeneic marrow transplantation is associated with low CD4 T cell counts. Am. J. Hematol. 54, 131–138 (1997).

    CAS  PubMed  Google Scholar 

  41. 41

    Jacobson, C.A. et al. Immune reconstitution after double umbilical cord blood stem cell transplantation: comparison with unrelated peripheral blood stem cell transplantation. Biol. Blood Marrow Transplant. 18, 565–574 (2012).

    CAS  PubMed  Google Scholar 

  42. 42

    Thomson, B.G. et al. Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation. Blood 96, 2703–2711 (2000).

    CAS  PubMed  Google Scholar 

  43. 43

    Gehre, N. et al. A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo. Eur. J. Immunol. 45, 932–942 (2015).

    CAS  PubMed  Google Scholar 

  44. 44

    Awong, G., La Motte-Mohs, R.N. & Zúñiga-Pflücker, J.C. Generation of pro-T cells in vitro: potential for immune reconstitution. Semin. Immunol. 19, 341–349 (2007).

    CAS  PubMed  Google Scholar 

  45. 45

    Nazareth, E.J.P., Rahman, N., Yin, T. & Zandstra, P.W. A multi-lineage screen reveals mTORC1 inhibition enhances human pluripotent stem cell mesendoderm and blood progenitor production. Stem Cell Reports 6, 679–691 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Aoyama, K. et al. The interaction of the Wnt and Notch pathways modulates natural killer versus T cell differentiation. Stem Cells 25, 2488–2497 (2007).

    CAS  PubMed  Google Scholar 

  47. 47

    Roozen, P.P.C., Brugman, M.H. & Staal, F.J.T. Differential requirements for Wnt and Notch signaling in hematopoietic versus thymic niches. Ann. NY Acad. Sci. 1266, 78–93 (2012).

    CAS  PubMed  Google Scholar 

  48. 48

    Huijskens, M.J.A.J. et al. Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells. J. Leukoc. Biol. 96, 1165–1175 (2014).

    PubMed  Google Scholar 

  49. 49

    Janas, M.L. et al. Thymic development beyond beta-selection requires phosphatidylinositol 3-kinase activation by CXCR4. J. Exp. Med. 207, 247–261 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Zakrzewski, J.L. et al. Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors. Nat. Biotechnol. 26, 453–461 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Holmes, R. & Zúñiga-Pflücker, J.C. The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro. Cold Spring Harb. Protoc. 2009, pdb.prot5156 (2009).

  52. 52

    Hsieh, J.J. et al. Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol. Cell. Biol. 16, 952–959 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank the donors and the Research Centre for Women's and Infants' Health BioBank of Mount Sinai Hospital for the human specimens used in this study and all the operators at the Sick Kids-University Health Network Flow Cytometry Facility for their technical support in cell sorting. The authors thank W. Wang and E. Piccinini for their assistance in processing fetal liver samples, T. Usenko for assistance in processing umbilical cord blood samples, J. Östblom for providing training in computational programming, C. Bauwens for assistance in editing the manuscript, J. Ma for illustrating the summary cartoon figure, and all members of the P.W.Z. laboratory for their helpful discussion. This work was supported by the Leukemia and Lymphoma Society of Canada (grant no. 493946 to P.W.Z.), the Canadian Institutes for Health Research (CIHR) (grant no. MOP-119538 to J.C.Z.-P.; grant no. 489401 and 452750 to P.W.Z.), Medicine by Design, a Canada First Research Excellence Program at the University of Toronto (grant no. C1TPA-2016-20 to J.C.Z.-P.; grant no. 499470 to P.W.Z.), and the Krembil Foundation (to J.C.Z.-P.). S.S. was supported by the CIHR Vanier Canada Graduate Scholarship and the NSERC CREATE M3 Scholarship. J.C.Z.-P. is the Canada Research Chair in Developmental Immunology. P.W.Z. is the Canada Research Chair in Stem Cell Bioengineering.

Author information

Affiliations

Authors

Contributions

S.S. designed and performed experiments, analyzed data, and wrote the manuscript. M.A.L. performed live imaging and PCR analyses. J.S. performed in vivo experiments. J.M.E. performed certain flow cytometry experiments. M.M. provided vital reagents and feedback. J.C.Z.-P. provided critical experimental advice and edited the manuscript. P.W.Z. designed experiments and edited the manuscript.

Corresponding author

Correspondence to Peter W Zandstra.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Representative flow cytometry plots on day 7 of differentiation in IMDM+BIT serum-free medium.

Flow cytometry plots depict differentiation in IMDM+BIT serum-free medium on 10 μg/mL adsorbed DL4 ligand. Data represents mean ± SD (n = 3).

Supplementary Figure 2 Quantification of progenitor cell expansion in IMDM+BIT or αMEM+BIT serum-free medium vs. OP9 serum medium control.

(a) Total fold expansion (cell yield on day 7 of CD45+7AAD live cells normalized to input sorted HSPCs on day 0) in the presence or absence of 10 μg/mL adsorbed DL4 in OP9 serum medium (αMEM+16% FBS) vs. serum-free media compositions (αMEM+BIT and IMDM+BIT) (n = 3). All media compositions contained the same amount of cytokines (25 ng/mL SCF, 5 ng/mL Flt3L and 1 ng/mL IL-7 in 200 μL medium/well) with a 50% medium exchange step at day 4. (b) CD11b+ myeloid cell expansion on day 7 normalized to input sorted HSPCs on day 0 in the different media compositions in the presence or absence of DL4 (n = 3). (c) CD19+ B cell expansion on day 7 normalized to input sorted HSPCs on day 0 in the different media compositions in the presence or absence of DL4 (n = 3). (d) CD25+CD90+ proT-cell expansion on day 7 normalized to input sorted HSPCs on day 0 in the different media compositions in the presence or absence of DL4 (n = 3). Data represent mean ± 95% CI. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary Figure 3 Quantification of frequency and yield of DN1, DN2 and DN3 proT-cell populations in IMDM+BIT or αMEM+BIT serum-free medium vs. OP9 serum medium control.

(a) Quantification of total CD45+7AAD live cell yield at day 7 vs. frequency of DN1 (CD25CD44+CD45+) proT-cells in OP9 serum medium (αMEM+16% FBS) vs. serum-free media compositions (αMEM+BIT and IMDM+BIT). (b) Quantification of yield of CD25+CD90+ proT-cells at day 7 vs. frequency of DN2 (CD25+CD44+CD45+) cells or (c) frequency of DN3 (CD25+CD44CD45+) cells in different serum-free media compositions compared to OP9 serum medium control. Shaded areas were plotted using a bivariate kernel density estimate function. All data points are depicted for n = 3 independent replicates.

Supplementary Figure 4 Optimization of key assay design parameter – Notch ligand choice.

(a) Quantification of DN1, DN2, DN3, CD19+ B cell, CD11b+ myeloid and CD25+CD90+ proT-cell subset frequencies on day 7 obtained on increasing coating concentrations of DL1 ligand (n = 3). (b) Notch pathway Cbf-1 Firefly activation normalized to constitutively active Renilla plasmid after 24 hours on 0 μg/mL DL4 (no ligand; negative control) and 10 μg/mL DL4 (positive control) to test activity of DL1 at 10 and 20 μg/mL (n = 3). Data represent mean ± 95% CI. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary Figure 5 Optimization of key assay design parameter - HSPC seeding density.

(a) Quantification of total CD45+7AAD live cell expansion at day 7 normalized to increasing input day 0 sorted HSPC seeding densities per cm2 (n = 3). (b) Quantification of DN1, DN2, DN3, CD19+ B cell, CD11b+ myeloid and CD25+CD90+ proT-cell subset frequencies on day 7 with increasing input day 0 sorted HSPC seeding densities per cm2 (n = 3). Data represent mean ± 95% CI. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary Figure 6 Optimization of key assay design parameter – well shape.

Quantification of DN1, DN2, DN3, CD19+ B cell, CD11b+ myeloid and CD25+CD90+ proT-cell subset frequencies at day 7 in U-bottom (U bot) vs. flat-bottom plates (flat bot) with no coating (- DL4) or 10 μg/ml DL4 (+ DL4) on day 0 (n = 3). Data represent mean ± 95% CI. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary Figure 7 Design of Experiment (DOE) in silico modeling to optimize cytokine concentrations and eliminate re-feeding.

(a) DOE response surface method was implemented to optimize the concentrations of SCF, Flt3L and IL-7 for the “no-feed” differentiation strategy. The design cube depicts the range of concentrations tested in the DOE model. (b) Schematic for elimination of day 4 medium exchange while reducing medium consumption. Baseline “re-feed” differentiation strategy involved seeding cells in 200 μL medium/well with 50% media exchange at day 4 with double the cytokine concentration at day 0 to maintain the same concentration. Our baseline control used 25 ng/mL SCF, 5 ng/mL Flt3L and 1 ng/mL IL-7 in 200μL medium/well (25-5-1 re-feed condition) based on previously published OP9 stromal co-culture. The optimized “no-feed” differentiation strategy involved seeding cells in 50 μL medium/well with higher cytokine concentrations and no medium exchange at day 4. (c) Surface response curve depicting desirability of varying SCF and IL-7 test concentrations at the optimal constant Flt3L concentration to maximize committed DN3 proT-cell frequency. (d) In vitro testing of the top predicted cytokine concentration hits from the in silico DOE model. Quantification of CD25+CD90+ proT-cell yield vs. DN2 frequency at day 7 with the positive control (25-5-1 re-feed condition) vs. the optimized no-feed process. By simply increasing the IL-7 concentration from 2 to 10 ng/mL (50-10-10 no-feed condition), the cells produced a significantly higher yield of DN2 and higher frequency and yield of T lineage-committed DN3 cells than the control (Fig. 1f). Shaded areas were plotted using a bivariate kernel density estimate function. All data points are depicted for n = 3 independent replicates.

Supplementary Figure 8 Increasing concentrations of VCAM-1 and not fibronectin enhance DN3 proT-cell commitment and yield.

(a) A screening study was performed to assess the effect of different concentrations of several cytokines (IL-6, soluble IL-6R (sIL6R), IL-11, IL-7, leukemia inhibitory factor (LIF)), chemokines (CCL25, SDF1α) and matrix protein (VCAM-1) on T-lineage committed DN3 cell frequency on day 7 of culture (n = 3). (b) Sorted HSPCs were cultured on 10 μg/mL DL4 alone or 10 μg/mL DL4 with increasing concentrations of fibronectin (0.5, 1.0 and 5.0 μg/mL). The concentrations of fibronectin are equivalent in molar concentrations to VCAM-1 tested in these dose titration experiments. DN1, DN2, DN3, CD19+ B cell, CD11b+ myeloid and CD25+CD90+ proT-cell frequencies were quantified on day 7 (n = 3). (c) Quantification on day 7 of total DN3 proT-cell yield on 10 μg/mL DL4 alone or 10 μg/mL DL4 with increasing concentrations of VCAM-1 (0.24, 0.47 and 2.32 μg/mL) (n = 4). Data represent mean ± 95% CI. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary Figure 9 Live imaging of differentiating proT-cells in the serum-free and feeder-free engineered thymic niche.

Representative still image from live imaging of differentiating day 7 proT-cells in the DL4+VCAM-1 engineered thymic niche. Cells were stained for CD25 (magenta) and CD44 (green) and merged with bright-field. Scale bar, 100μm.

Supplementary Figure 10 VCAM-1 synergizes with DL4 to enhance proT-cell differentiation and Notch pathway activation.

(a) Sorted HSPCs were cultured on sub-optimal 5 μg/mL DL4 alone or 5 μg/mL DL4 with increasing concentrations of VCAM-1 (0.24, 0.47 and 2.32 μg/mL). DN1, DN2, DN3, CD19+ B cell, CD11b+ myeloid and CD25+CD90+ proT-cell frequencies were quantified on day 7 (n = 3). (b) Notch pathway Cbf-1 Firefly activation normalized to constitutively active Renilla plasmid after 24 hours on no ligand (negative control), 2.32 μg/mL VCAM-1, 10 μg/mL DL4 (positive control) and 10 μg/mL DL4 with 2.32 μg/mL VCAM-1 (n = 4). Data represent mean ± 95% CI. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary Figure 11 Rapid DN2 and DN3 proT-cell differentiation within first two days of culture on DL4+VCAM-1 than DL4 alone.

Representative flow plots of HSPCs 24 and 48 hours after culture on DL4 vs. DL4+VCAM-1 (n = 4).

Supplementary Figure 12 Input CD34+ frequency of human HSPCs.

The purity of the input umbilical cord blood-derived HSPCs was verified to be greater than 95% CD34+ prior to initiation of each culture. CD34+ frequency was assessed on 7AAD live cell population (n = 3).

Supplementary Figure 13 qRT-PCR gene expression in human HSPCs after 24 hours of culture.

qRT-PCR gene expression of downstream Notch pathway genes (HES1, DELTEX, NOTCH1, BCL11B, GATA3, TCF7), a HSPC gene (E2A) and a myeloid lineage gene (PU.1) on no coating, 2.32 μg/mL VCAM-1, 10 μg/mL DL4, or DL4+VCAM-1 after 24 hours of culture with human CD34+ HSPCs (n = 5). Data represent mean ± SE. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary Figure 14 Comparison of day 14 proT-cell frequencies from OP9-DL4 co-culture or DL4+VCAM-1 engineered thymic niche.

Quantification of CD7+CD34+, CD7+CD34 and CD7+CD5+ populations after 14 days of OP9-DL4 co-culture or on the engineered thymic niche (n = 6). Data represent mean ± 95% CI. * P < 0.05; ** P < 0.01; *** P < 0.001.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Tables 1–32 (PDF 2609 kb)

Live cell time-lapse imaging of proT-cells on DL4

Transmitted light, 10-minute frame interval. (MOV 5149 kb)

Live cell time-lapse imaging of proT-cells on DL4+VCAM-1

Transmitted light, 10-minute frame interval. (MOV 5754 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shukla, S., Langley, M., Singh, J. et al. Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1. Nat Methods 14, 531–538 (2017). https://doi.org/10.1038/nmeth.4258

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing