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Research in the Kaufman Lab


Research Areas

Development of Functional Blood cells from human ESCs and iPSCs

Development of Functional Blood cells from human ESCs and iPSCs

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).

Clinical strategy for wide-scale human iPSC-based gene and cell therapy

Clinical strategy for wide-scale human iPSC-based gene and cell therapy

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).

Engineering iPSC-Derived NK Cells to Improve Anti-Cancer Activity

The main focus in our research group is to use human pluripotent stem cells as a platform to derive improved cell-based therapies. We have now demonstrated that human induced pluripotent stem cells (iPSCs) that have been engineered on the stem cell-level can routinely produce iPSC-derived natural killer (NK) cells that uniformly express genetic modifications. This technique allows production of iPSC-derived NK cells that express proteins such as chimeric antigen receptors (CARs) and cytokine signaling receptors or with target genes disrupted ("knocked-out").

Recent publications from our group highlight the power of these approaches. First, we demonstrated iPSC-NK cells that express NK cell-optimized CARs were shown to mediate strong antigen-specific NK cell activation. These CAR-NK cells demonstrated increased anti-tumor killing against both solid tumors and hematologic malignancies (Li et al., 2018). A second seriews of studies showed that expression of a non-cleavable CD16 Fc-receptor in iPSC-NK cells mediated increased antibody-dependent cellular cytotoxicity against a range of target tumors. The combination of these engineered NK cells with monoclonal antibodies led to increased killing of tumors that are more resistant to NK cell killing (Zhu et al., 2020). Additionally, we demonstrated that deletion of internal genes can also improve the function of iPSC-derived NK cells. Knockout of the gene CISH, a negative regulator of cytokine signaling, in iPSC-NK cells was shown to increase NK cell expansion and killing of tumors. This approach demonstrated that disruption of genes involved in NK cell signaling and metabolism can generate NK cells with increased anti-tumor activity (Zhu et al., 2020).

Ongoing efforts are testing new CAR strategies and other genetic modifications to build newer, improved iPSC-derived NK cells.

Derivation of Macrophages from Human Pluripotent Stem Cells


Derivation of Macrophages from Human Pluripotent Stem Cells

Studies from our group also demonstrate that it is very efficient to derive macrophages from human pluripotent stem cells. We can also polarize these iPSC-derived macrophages to M1 and M2-type macrophages. M1 macrophages have pro-inflammatory, anti-tumor activity, whereas M2 macrophages have anti-inflammatory activity that can help mediate tissue repair and regeneration. Ongoing studies are developing macrophage-specific CARs to derive iPSC-CAR-macrophages to better target more resistant solid-tumor malignancies. We have also demonstrated iPSC-macrophages can mediate repair in a xenograft model of hepatic fibrosis.

CRISPR Screening Methods to Identify Novel Mechanisms that Regulate NK Cell-Mediated Activity


CRISPR screening methods to identify novel mechanisms that regulate NK cell-mediated activity

Inside a solid tumor the selective pressure pushes tumor cells to develop resistance mechanisms to escape NK cell-mediated killing. Recently with the improvement of the genome editing using the CRISPR/Cas9 method it is possible to study and identify new genes that are involved in tumor cell proliferation, drug resistance and metastasis of cancer cells. CRISPR libraries were applied to screenings with genome-wide loss- or gain-of-functions. Studies in our group are utilizing a novel strategy using a "two cell type" CRISPR/Cas9 screening to identify mechanisms of resistance or sensitivity that will mediate better targeting tumor cells by NK cells. We identified a panel of potential genes that are essential in tumor cells for survival under selective pressure of NK cells. This strategy will allow us to test pharmacological and genetic strategies to up or down-regulate molecular targets on tumor cells that could be translated into clinical trials to improve NK cell-mediated immunotherapy.

Anti-tumor activities of CAR-targeted iPSC-derived NK cells

Anti-tumor activities of CAR-targeted iPSC-derived NK cells

Improved Tumor Killing by hESC/iPSC-derived NK cells
Improved Tumor Killing by hESC/iPSC-derived NK cells

Figure from Woll, 2009 & Hermanson, 2016

CSC 2018
Figure from Kaufman, 2018


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.

Bernareggi D, Pouyanfard S, Kaufman DS. (2019). Development of innate immune cells from human pluripotent stem cells. Exp Hematol. 71:13-23.

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.

Li Y, Hermanson DL, Moriarity BS, 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.

Zhu H, Lai YS, Li Y, Blum R, Kaufman DS. (2018). Concise Review: Human Pluripotent Stem Cells to Produce Cell-based Cancer Immunotherapy. Stem Cells. 36(2):134-145.

Zhu H, Blum R, Bjordahl R, Gaidarova S, Rogers P, Lee TT, Abujarour R, Bonello GB, Wu J, Tsai PF, Miller JS, Walcheck B, Valamehr B, Kaufman DS. (2020). Pluripotent stem cell-derived NK cells with high-affinity non-cleavable CD16a mediate improved anti-tumor activity. Blood. 135(6):399-410.

Zhu H, Blum R, Bernareggi D, Ask EH, Wu Z, Hoel HJ, Meng Z, Wu C, Guan KL, Malmberg KJ, Kaufman DS. (2020). Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity. Cell Stem Cell. 27(2):224-237.

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