Figure from Kaufman, 2009 Studies in the Kaufman Lab focus on use of human pluripotent stem cells to study basic mechanisms that regulate early human blood cell development and to derive therapeutic mature blood cell populations
. These studies utilize both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). Both hESCs and iPSCs can be maintained in long-term culture with stable karyotype and without loss of the developmental potential to make all the cells and tissues in the body. Therefore hESCs/iPSCs provide an ideal platform both for studies of human hematopoiesis and large-scale production of blood cells such as lymphocytes that can be used for novel therapies against cancer and other diseases. (Angelos and Kaufman, 2015; Kaufman, 2009)
Figure from Angelos & Kaufman, 2015
Early hematopoietic development from human pluripotent stem cells:
One key goal since hESCs and iPSCs were first isolated or produced has been to use these pluripotent stem cells as a source for in vitro-derived hematopoietic stem cells (HSCs) that can be used for blood and marrow transplantation (BMT). Studies in the Kaufman Lab were the first to produce blood cells from hESCs (Kaufman et al., 2001). However, we are still unable to produce putative HSCs capable of long-term, multi-lineage engraftment when transplanted info immunodeficient mice. We continue to pursue this goal and have engineered hESCs and iPSCs in various ways to improve putative HSC development. These approaches include expression of luciferase to allow monitoring of blood cells in vivo by bioluminescent imaging (Tian et al., 2009; Tian et al., 2006), expression of reporter constructs for key regulators of early hematopoiesis such as RUNX1(Ferrell et al., 2015), and engineering gene expression such as the aryl hydrocarbon receptor (AHR) that regulate early human hematopoiesis (Angelos et al., 2017).
Figure from Angelos, 2017
Figure from Ferrell, 2015 Production of lymphocytes from human pluripotent stem cells:
A second key goal of the Kaufman lab is to produce immune cells from hESCs/iPSCs suitable to treat refractory cancers or potentially other diseases. Our most successful results have been to derive natural killer (NK) cells from hESCs and iPSCs (Hermanson et al., 2016; Knorr et al., 2013; Ni et al., 2011; Woll et al., 2009). NK cells are normal part of the innate immune system capable of killing certain tumors and virally-infected cells Clinical studies for over a decade have used NK cells isolated from peripheral blood of allogeneic donors for adoptive transfer to treat patients, mainly with relapsed or refractory acute myelogenous leukemia (AML). However, these NK cells must be isolated from individual donors on a patient-specific basis. NK cells derived from hESCs and iPSCs have a receptor profile and gene expression signature similar to NK cells isolated from peripheral blood or isolated from human umbilical cord blood. hESC/iPSC-NK cells can also kill diverse tumors both in vitro and in vivo.
Figure from Woll, 2009 & Hermansan, 2016
Figure from Kaufman, 2018
We are now pursuing several strategies to move hESC/iPSC-derived NK cells into clinical therapies:
Large scale production of unmodified hESC/iPSCs-NK cells to treat hematological malignancies (blood cell cancers). Our studies demonstrate that not only can we produce NK cells with anti-tumor activity from hESCs/iPSCs, we can also routinely produce clinical-scale quantities of these NK cells in defined serum-free, xeno-free culture conditions (Knorr et al., 2013). These unmodified hESC/iPSC-NK cells are suitable for treatment of AML, multiple myeloma, and potentially other hematological malignancies. Addition, since we can analyze the repertoire of killer immunoglobulin-like receptors (KIRs) of the hESCs and iPSCs, we can produce NK cells with optimized KIR expression for treatment of patients with differing HLA haplotypes.
Production of iPSC-NK cells with chimeric antigen receptors (CARs) to target refractory tumors. CAR-expressing T cells have revolutionized cell-based therapies and are now FDA-approved for treatment of CD19-expressing tumors such as acute lymphocytic leukemia (ALL) and lymphomas. However, the current CAR-T cell strategy requires isolation and gene modification of T cells to be done on a patient-specific basis. Additionally, treatment of solid tumors with CAR-T cells has been less successful than targeting CD19-expressing tumors. We have now demonstrated that human iPSCs can be engineered on the stem cell-level to routinely produce iPSC-derived CAR-expressing NK cells. We are currently testing these cells against solid tumors such as ovarian cancer. We are also testing novel CAR signaling domains to optimize this strategy.
This approach allows for a standardize, off-the-shelf, targeted immunotherapy against both relapsed/refractory solid tumors and hematological malignancies(Kaufman, 2018).
Derivation of other blood cells and non-blood cells from hESCs and iPSCs.
Production of hESC/iPSC NK cells with improved expression of CD16 to mediate more effective antibody-dependent cell-mediated cytotoxicity (ADCC). The Fcγ receptor CD16a is a key mediator of NK cell activity by binding the Fc portion of antibodies leading activation of intracellular signaling pathways to mediate ADCC. Indeed, NK cell-mediated ADCC plays a key role in anti-tumor activity of patients treated with targeted anti-cancer antibodies such as rituximab (against CD20-expressing hematological malignancies) and cetuximab (against EGFR-expressing solid tumors). However, both tumors and activated NK cells can express the metalloprotease ADAM17 that cleaves CD16 (and other proteins) from the surface of NK cells, leading to loss of ADCC. Our collaborator designed a non-cleavable version of CD16a with a single residue (197) mutated from a serine to proline to prevent ADAM17-mediated CD16 cleavage(Jing et al., 2015). We have now used our strategy to derive NK cells from hESCs/iPSCs to stably integrate a high affinity, cleavage-resistant CD16 molecule into human iPSCs that can then be used to produce NK cells that maintain improved CD16 expression and more potent anti-tumor activity. We are also testing other strategies to improve CD16 expression.
Studies in the Kaufman lab also pursue derivation and therapeutic translation of other cell populations from hESCs and iPSCs. Currently, this work includes optimizing strategies for macrophage/monocyte production and improved T cell development. Previous studies have also demonstrated effective production of endothelial cells and other vascular cells that can be used for cardiovascular repair(Ye et al., 2014). We have also derived osteogenic (bone forming) cells from hESCs and iPSCs that are suitable for treatment of non-healing bone fractures and other orthopedic conditions(Zou et al., 2016; Zou et al., 2015). KEY REFERENCES:
Angelos, M.G., and Kaufman, D.S. (2015). Pluripotent stem cell applications for regenerative medicine. Curr Opin Organ Transplant 20, 663-670.
Angelos, M.G., Ruh, P.N., Webber, B.R., Blum, R.H., Ryan, C.D., Bendzick, L., Shim, S., Yingst, A.M., Tufa, D.M., Verneris, M.R., et al. (2017). Aryl hydrocarbon receptor inhibition promotes hematolymphoid development from human pluripotent stem cells. Blood 129, 3428-3439.
Ferrell, P.I., Xi, J., Ma, C., Adlakha, M., and Kaufman, D.S. (2015). The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells. Stem Cells 33, 1130-1141.
Hermanson, D.L., Bendzick, L., Pribyl, L., McCullar, V., Vogel, R.I., Miller, J.S., Geller, M.A., and Kaufman, D.S. (2016). Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer. Stem Cells 34, 93-101.
Hermanson, D.L., and Kaufman, D.S. (2015). Utilizing chimeric antigen receptors to direct natural killer cell activity. Frontiers in immunology 6, 195.
Jing, Y., Ni, Z., Wu, J., Higgins, L.A., Markowski, T.W., Kaufman, D.S., and Walcheck, B. (2015). Identification of an ADAM17 Cleavage Region in Human CD16 (FcγRIII) and the Engineering of a Non-Cleavable Version of the Receptor in NK Cells. PLoS ONE, e0121788.
Kaufman, D.S. (2009). Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells. Blood 114, 3513-3523.
Kaufman, D.S., Hanson, E.T., Lewis, R.L., Auerbach, R., and Thomson, J.A. (2001). Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 98, 10716-10721.
Kaufman DS. (2018) Human iPSC-derived Natural Killer Cells Engineered with Chimeric Antigen Receptors
Enhance Anti-Tumor Activity. Cell Stem Cell. 23(2), 181-192.
Knorr, D.A., Ni, Z., Hermanson, D., Hexum, M.K., Bendzick, L., Cooper, L.J.N., Lee, D.A., and Kaufman, D.S. (2013). Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Translational Medicine 2, 274-283.
Ni, Z., Knorr, D.A., Bendzick, L., Allred, J., and Kaufman, D.S. (2014). Expression of chimeric receptor CD4ζ by natural killer cells derived from human pluripotent stem cells improves in vitro activity but does not enhance suppression of HIV infection in vivo. Stem Cells 32, 1021-1031.
Ni, Z., Knorr, D.A., Clouser, C.L., Hexum, M.K., Southern, P., Mansky, L.M., Park, I.-H., and Kaufman, D.S. (2011). Human pluripotent stem cells produce natural killer cells that mediate anti-HIV-1 activity by utilizing diverse cellular mechanisms. Journal of Virology 85, 43-50.
Tian, X., Hexum, M.K., Penchev, V.R., Taylor, R.J., Shultz, L.D., and Kaufman, D.S. (2009). Bioluminescent imaging demonstrates that transplanted human embryonic stem cell-derived CD34(+) cells preferentially develop into endothelial cells. Stem cells 27, 2675-2685.
Tian, X., Woll, P.S., Morris, J.K., Linehan, J.L., and Kaufman, D.S. (2006). Hematopoietic Engraftment of Human Embryonic Stem Cell-Derived Cells Is Regulated by Recipient Innate Immunity. Stem Cells 24, 1370-1380.
Woll, P.S., Grzywacz, B., Tian, X., Marcus, R.K., Knorr, D.A., Verneris, M.R., and Kaufman, D.S. (2009). Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity. Blood 113, 6094-6101.
Ye, L., Chang, Y.-H., Xiong, Q., Zhang, P., Zhang, L., Somasundaram, P., Lepley, M., Swingen, C., Su, L., Wendel, J.S., et al. (2014). Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells. Cell Stem Cell 15, 750-761.
Zou, L., Chen, Q., Quanbeck, Z., Bechtold, J.E., and Kaufman, D.S. (2016). Angiogenic activity mediates bone repair from human pluripotent stem cell-derived osteogenic cells. Sci Rep 6, 22868.
Zou, L., Kidwai, F.K., Kopher, R.A., Motl, J., Kellum, C.A., Westendorf, J.J., and Kaufman, D.S. (2015). Use of RUNX2 expression to identify osteogenic progenitor cells derived from human embryonic stem cells. Stem Cell Reports 4, 190-198.