Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells

Abstract

Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.

Figure 1

Serum- and cytokine-free EC cocultures supplemented with low doses of SCF, Flt3L, and TPO expand phenotypically marked HPCs/HSPCs and differentiated hematopoietic progenitors. Human umbilical cord blood (hCB) was enriched for CD34⁺ cells, and 5 × 10⁴ CD34⁺ cells were cocultured with or without E4⁺ECs. (A-C) Cocultured hematopoietic cells were cumulatively expanded. Day 12 was chosen for analyses because of rapid attrition of hematopoietic cells, which were cultured without feeder cells. (A) Total hematopoietic cell expansion. (B) Total CD45⁺CD34⁺ HPC expansion. (C) Total phenotypically marked HSPC expansion defined by CD45⁺ Lin⁻CD34^hi+CD45RA⁻CD49f⁺. Note that human hematopoietic cells that were cocultured with E4⁺ECs expanded significantly more compared with cultures without vascular feeder cells. (D) Representative contour plots of day 12 cumulative expansion of CD45⁺Lin⁻CD34^hi+CD45RA⁻CD49f⁺ on E4⁺ECs. (E) Representative contour plots of day 12 cumulative expansion of CD33⁺CD14⁺ and CD33⁺CD15⁺ myeloid progenitors on E4⁺ECs. (F) Representative contour plots of day 12 cumulative expansion of CD51/61⁺CD41a⁺CD42b⁺ megakaryocytic progenitor cells on E4⁺ECs. Ranges of fold expansion are listed in supplemental Table 1. Results were analyzed using Student t test, and P < .05 was considered significant. Data are mean ± SD.

Figure 2

Human CD34⁺ CBs cocultured with ECs give rise to multilineage engraftment in NSG mice. (A) Percentage of engrafted human CD45⁺ hematopoietic cells at 3 and 9 weeks after transplantation. (B) Total number of engrafted phenotypically marked human HPCs. (C) Total number of terminally differentiated mature cells per femur. (D) Percentage of human CD45 engraftment in secondary recipients transplanted with human CD45⁺ hematopoietic cells from primary recipients in panels A through C (N = 10 for each condition; 9 of 10 in the E4⁺EC group and 5 of 10 in the cytokines-alone group had significant human CD45 engraftment: > 1%). All human cells analyzed with the cell population that was gated on cells that were negative for mouse Ter119 and CD45. (E) A total of 50 000 CD45⁺CD34⁺ hCB cells were cultured with or without feeder layers in serum-free conditions and supplemented with 50 ng/mL SCF, 50 ng/mL TPO, and 50 ng/mL Flt3L for 12 days. CD45⁺CD34⁺ cells were isolated and transplanted into NSG mice with the corresponding cell doses. Unmanipulated CD45⁺CD34⁺ were used as controls. At 3 and 9 weeks after transplantation, mice were analyzed for human CD45⁺ cells in bone marrow. Mice with > 1% engraftment were considered positive. Mice that were positive at 3 weeks remained positive at 9 weeks, with higher levels of human CD45⁺ engraftment at 9 weeks. SCID-repopulating cells were determined using L-Calc Version 1.1 software. ***P = .0001. *P = .05. Reference group: E4⁺ECs + cytokines, analyzed 9 weeks after transplantation. (F) Representative contour plots of human HPC engraftment 9 weeks after transplantation. (G) Representative contour plots of multilineage human hematopoietic cell engraftment 9 weeks after transplantation. Note that human hematopoietic cells cocultured with E4⁺ECs have greater engraftment potential and give rise to a higher number of multilineage human hematopoietic progenitors and long-term engraftment in secondary recipients. Results were analyzed using the Student t test, and P < .05 was considered significant. Date are mean ± SD.