This invention is directed to, inter alia, compounds, methods, systems, and compositions for the maintenance, enhancement, and expansion of hematopoietic stem cells derived from one or more sources of CD34+ cells. Sources of CD34+ cells include bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood. Also provided herein are compounds of Formula I ##STR00001## which are useful in maintaining, enhancing, and expanding of hematopoietic stem cells.

Provided herein are compositions and methods for preparing T cell compositions and uses thereof, including methods for treating cancer in a subject in need thereof by administering T cells induced with peptides comprising an epitope sequence from a library of epitope sequences, wherein each epitope sequence in the library is matched to a protein encoded by an HLA allele and binds to a protein encoded by an HLA allele of the subject, is immunogenic according to an immunogenic assay, is presented by antigen presenting cells according to a mass spectrometry assay, and stimulates T cells to be cytotoxic according to a cytotoxicity assay.

This invention concerns chemically defined haematopoietic induction media that support the differentiation of haemogenic endothelial cells (HECs) into haematopoietic progenitor cells (MFCs) that are capable of further differentiation into T cells. The media may (a) stimulate cKIT receptor and/or cKIT receptor mediated signalling pathways and/or (b) stimulate VEGFR and/or VEGFR mediated signalling pathways. For example, the medium may comprise SCF and VEGF. In some embodiments, the media may further (c) stimulate MPL or MPL mediated signalling pathways; (d) stimulate FLT3 or FLT3 mediated signalling pathways (e) stimulate IGF1R or IGF1R mediated signalling pathways and (f) display interleukin (IL) activity. For example, the medium may further comprise Thrombopoietin (TPO), Fits ligand (FltSL), IGF-1, IL-3, IL-6 and optionally IL-7. These media may be useful for example in the production of blood cells or use in immunotherapy.

The present disclosure relates to methods for expanding populations of hematopoietic stem cells (HSCs) using a genetically engineered human fetal liver niche and compositions of purified ex vivo expanded HSCs. Also provided herein are methods of using such expanded HSC cell populations for clinical applications including allogeneic hematopoietic stem cell transplantation and for drug discovery and modeling human liver development.

The present invention provides NK cell compositions and/or preparations and methods of using such NK cell compositions and/or preparations for immunotherapy. The NK cell compositions and/or preparations can be used in therapies of a broad range of viral infections, bacterial infections, cancer and leukemia malignancies, and other diseases.

Aspects of the present disclosure relate to methods and compositions related to the preparation of immune cells, including engineered immune cells. Certain embodiments of the disclosure include compositions, cells, and methods related to engineered invariant natural killer T (iNKT) cells for off-the-shelf use for clinical therapy. The iNKT cells may be produced from hematopoietic stem progenitor cells and may be suitable for allogeneic cellular therapy because they are HLA negative. In some aspects, the cells have imaging and suicide targeting capabilities.

The present invention relates to, inter alia, an engineered cell (e.g., iPSC, IPS-derived NK, or NK cell) comprising a disrupted B2M gene and an inserted polynucleotide encoding one or more of SERPINB9, a fusion of IL15 and IL15Rα, and/or HLA-E. The engineered cell can further comprise a disrupted CIITA gene and an inserted polynucleotide encoding a CAR, wherein the CAR can be an anti-BCMA CAR or an anti-CD30 CAR. The engineered cell may further comprise a disrupted ADAM17 gene, a disrupted FAS gene, a disrupted CISH gene, and/or a disrupted REGNASE-1 gene. Methods for producing the engineered cells are also provided, and therapeutic uses of the engineered cells are also described. Guide RNA sequences targeting described target sequences are also described.

A method of producing a collection of natural killer cells from CD34.sup.+ human stem cells. The invention further provides to a collection of natural killer cells thus produced and a pharmaceutical composition having such, natural killer cells. Further, the invention relates to a method of using the pharmaceutical composition as a medicament, in particular for immunotherapy in the treatment of malignancies.

The present disclosure relates to a method of culturing quiescent hematopoietic stem cells. This method involves providing a culture medium and introducing, into the culture medium, quiescent hematopoietic stem cells to culture the stem cells and maintain quiescence of the stem cells. The culture medium comprises a vacuolar-H.sup.+ adenosine triphosphate ATPase (“v-ATPase”) inhibitor. Also disclosed are methods of treating a subject for a hematological disorder, methods of culturing leukemic stem cells, and methods of enhancing the hematopoietic reconstitution ability of a population of human hematopoietic stem cells.

Aspects of the present disclosure relate to methods and compositions related to the preparation of immune cells, including engineered T cells comprising at least one exogenous γδ T cell receptor, for example one that is selected to target a specific disease or pathogen (e.g., cancer or COVID-19). The T cells may be produced from human hematopoietic stem/progenitor cells and are suitable for allogeneic cellular therapy because they do not induce graft-versus-host disease (GvHD) and resist host immune allorejection. Consequently, such cells are suitable for off-the-shelf use in clinical therapy.