Methods of preparing hematopoietic progenitor cells in vitro
Inventors
Vizcardo, Raul E. • Tamaoki, Naritaka • Good, Meghan L. • Restifo, Nicholas P.
Assignees
US Department of Health and Human Services
Publication Number
US-12037607-B2
Publication Date
2024-07-16
Expiration Date
2038-11-08
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Abstract
Disclosed are methods of preparing CD34+CD43+ hematopoietic progenitor cells (HPC) in vitro according to embodiments of the invention. Also disclosed are methods of differentiating CD34+CD43+ hematopoietic progenitor cells to hematopoietic lineage cells according to embodiments of the invention. Also disclosed are methods of treating or preventing a condition in a mammal, e.g., cancer, according to embodiments of the invention.
Core Innovation
The invention provides methods of preparing CD34+CD43+ hematopoietic progenitor cells (HPC) in vitro using multi-step three-dimensional (3D) culture techniques. The methods involve culturing source cells, preferably human induced pluripotent stem cells (iPSC), forming embryoid bodies (EBs) in a first 3D culture, mixing the EBs with mesoderm lineage cells (MLC) to form a single cell suspension, and co-culturing the mixture in a second 3D culture to form hematopoietic spheroids. These spheroids are then cultured further in a third 3D culture to differentiate and harvest CD34+CD43+ HPC.
The invention also provides alternative methods of forming spheroids directly from source cells in a bioreactor without first forming EBs, followed by mixing with MLC, co-culturing to generate hematopoietic spheroids, and harvesting CD34+CD43+ HPC. Additionally, the methods include differentiating these HPC into hematopoietic lineage cells, such as CD4+CD8+ T cells, by co-culturing with OP9-DL1 mouse stromal cells or other techniques that induce Notch signaling.
The problem solved by the invention arises from difficulties in producing multipotential HPC in vitro for adoptive cell therapy (ACT) applications. Existing 2D culturing methods, especially those using human mesenchymal stem cells (hMSC) as feeder cells, demonstrated poor and inconsistent production of CD34+CD43+ HPC and failed to generate functional T cells. The invention addresses the need for improved materials and methods to reliably and effectively prepare HPC suitable for therapeutic use.
Claims Coverage
The patent includes one independent claim that defines a multi-step in vitro method for preparing human CD34+CD43+ hematopoietic progenitor cells (HPC) with detailed culturing conditions and compositions.
Stepwise 3D culturing to form HPC
The method comprises culturing human source cells, preparing a single cell suspension, forming embryoid bodies (EBs) in a first 3D culture, mixing EBs with human mesoderm lineage cells (MLC) to prepare a suspension, co-culturing in a second 3D culture without exogenous cytokines to form hematopoietic spheroids, culturing the spheroids in a third 3D culture, and harvesting CD34+CD43+ HPC.
Versatile 3D culture formats
One or more of the first, second, and third 3D cultures can be performed in hanging drop cultures, 3D microwell cultures, hydrophobic surfaces, rotational or static 3D suspension cultures, or bioreactors.
Use of xeno-free media containing human platelet lysate
The method includes culturing hematopoietic spheroids in xeno-free medium, preferentially containing human platelet lysate.
Flexibility of cytokine conditions
The method may be carried out in the absence or presence of one or more exogenous cytokines, including a specified list such as FLT3L, TPO, VEGF, PDGF, bFGF, BMP4, IL-3, IL-6, SCF, G-CSF, GM-CSF, M-CSF, interferons, TNFs, WNT activators, ascorbic acid, and mono-thioglycerol.
Reprogramming human T cells to iPSC as source cells
The source cells used in the method can be induced pluripotent stem cells (iPSC) produced by reprogramming human T cells, or other pluripotent or lineage-reprogrammed cells.
Differentiation of HPC to CD4+CD8+ cells
The method includes further differentiating harvested CD34+CD43+ HPC to CD4+CD8+ T cells.
The independent claim covers a comprehensive multi-stage 3D culturing method for producing human CD34+CD43+ hematopoietic progenitor cells, specifying culture vessels, cell types, media conditions including xeno-free and cytokine presence/absence, and differentiation steps, thereby providing flexible and scalable approaches for generating HPC.
Stated Advantages
The methods allow for mass production of HPC suitable for clinical applications such as adoptive cell therapy.
The invention provides a fully autologous system that reduces or eliminates the need for invasive procedures on patients.
Use of frozen hMSC saves time and manpower compared to continuous culturing of fresh hMSC.
The 3D culture conditions improve the efficiency and reproducibility of HPC production compared to 2D cultures.
Culturing in xeno-free media containing human platelet lysate supports clinical compatibility and reduces contamination risks.
Documented Applications
Use of CD34+CD43+ HPC and their differentiated hematopoietic lineage cells for treating or preventing diseases including cancer, immunodeficiencies, autoimmune conditions, infections, and blood conditions.
Generation of blood products of rare blood types for blood banking and treatment of anemias and cytopenias.
In vitro generation of various hematopoietic lineage cells and sub-products such as human serum, antibodies, and cytokines.
Generation of an immune system for patients with immunodeficiencies and immune system reconstitution post irradiation or chemotherapy.
Generation of T cells with T-cell receptors specific for cancer or other diseases.
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