Genetically engineered human fetal liver niche as platform for biomanufacturing of hematopoietic stem cells
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.
1 . A method for ex vivo expansion of human hematopoietic stem cells (HSCs), the method comprising
(a) contacting a cell population comprising the human HSCs with a synthetic fetal liver organoid comprising mesenchymal-like cells, CD34-expressing (CD34+) endothelial-like cells, desmin-expressing (DES+) stellate-like cells, and CEBPα+ hepatocyte-like cells, wherein at least a portion of cells of the synthetic fetal liver organoid are genetically modified to express one or more of Stem Cell Factor (SCF), FMS-like Tyrosine Kinase 3 Ligand (FLT3L), and Thrombopoietin (TPO); and
(b) culturing the contacted organoid under conditions that promote proliferation of the human HSCs for about 3 to about 10 days, whereby an expanded population comprising CD34+ human HSCs is obtained.
2 . The method of claim 1 , wherein the expanded population comprises at least 3-fold more CD34+ HSCs than the cell population of step (a).
3 . The method of claim 1 , further comprising obtaining the cell population of step (a) from a human subject.
4 . The method of claim 1 , wherein the cell population comprising the HSCs is derived from umbilical cord blood or bone marrow.
5 . The method of claim 1 , wherein the synthetic fetal liver organoid constitutes a coating on a solid carrier.
6 . The method of claim 5 , wherein the carrier is a particulate support.
7 . The method of claim 1 , further comprising administering the expanded population of CD34+ human HSCs to a subject from whom the original cell population comprising HSCs is obtained.
8 . The method of claim 1 , wherein the contacted organoid is cultured in a medium selected from IMDM medium and APEL medium.
9 . The method of claim 8 , wherein the culture medium is supplemented with one or more of Stem Cell Factor (SCF), FMS-like Tyrosine Kinase 3 Ligand (FLT3L), and Thrombopoietin (TPO).
10 . The method of claim 1 , wherein the synthetic fetal liver organoid is obtained by:
(i) introducing into human pluripotent stem cells (hPSCs) one or more vectors comprising an inducible transgene encoding GATA-binding protein 6 (GATA6);
(ii) inducing expression of the GATA6 transgene in the hPSCs;
(iii) culturing the induced hPSCs in the presence of a pluripotency supporting medium for about 5 days, whereby a cell population comprising at least 70% CXCR4 + cells is obtained; and
(iv) culturing the cell population of step (iii) in a basal cell culture medium for about 10 days, whereby a synthetic, vascularized fetal liver organoid comprising CD34 + endothelial-like cells, NES + mesenchymal stem cell-like cells, DES + stellate-like cells, and CEBPα + hepatocyte-like cells is obtained.
11 . The method of claim 10 , wherein the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.
12 . The method of claim 1 , wherein the synthetic fetal liver organoid exhibits one or more properties selected from (i) an interconnected vasculature; (ii) differentiated cells within the mature liver organoid mutually contact each other in three dimensions; and (iii) more than one layer of cells.
13 . A method for ex vivo expansion of human hematopoietic stem cells (HSCs), the method comprising
(a) contacting a cell population comprising the human HSCs to a synthetic fetal liver organoid comprising mesenchymal-like cells, CD34-expressing (CD34+) endothelial-like cells, desmin-expressing (DES+) stellate-like cells, and CEBPα+ hepatocyte-like cells, wherein the synthetic fetal liver organoid is derived from embryonic stem cells (ESCs) or human pluripotent stem cells (hPSCs) engineered to express GATA 6 , wherein the cell population comprising the human HSCs are not derived from the engineered ESCs or hPSCs; and
(b) culturing the contacted organoid under conditions that promote proliferation of the human HSCs for about 3 to about 10 days, whereby an expanded population comprising CD 34 + human HSCs is obtained.
14 . The method of claim 13 , wherein before contacting the human HSCs with the fetal liver organoid, the method comprises isolating the human HSCs from a population of cells obtained from a tissue sample, umbilical cord blood, or bone marrow.
15 . The method of claim 13 , wherein the human HSCs are exogenous human HSCs.