Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation

Abstract

Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here, we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation.

Figure 1

Culture of total human cord blood cells in the presence of Angptl5 stimulates ex vivo expansion of HSCs. (A) Culture of 2.5 × 10⁷ total human cord blood cells was initiated in serum-free STIF medium or the same STIF medium containing 100 ng/mL Angptl5, and total cell numbers were counted at the indicated time. (B) Amount of human chimerism in the bone marrow of NOD/SCID mice that received a transplant of 10⁶ uncultured human mononuclear cord blood cells or the cultured progeny of 10⁶ initial human cord blood cells. Each symbol represents the engraftment of a single mouse that underwent transplantation assayed at 2 months after transplantation. *Significantly different from lane 1 value. Student t test, P < .001.

Figure 2

Culture of human cord blood CD34⁺ cells in the presence of Angptl5 stimulates ex vivo expansion of SRCs. (A) Culture of 10⁴ fresh human cord blood CD34⁺ cells was initiated in serum-free STF medium or STF medium supplemented with 500 ng/mL human Angptl5. Total cell numbers were counted. (B) Extent of human chimerism in the bone marrow of NOD/SCID mice that received a transplant of 10 000 fresh human cord blood CD34⁺ cells, or the progenies of initial 5000 cells cultured for 10 days in STF medium or STF medium supplemented with Angptl5. Each symbol represents the engraftment of a single mouse that underwent transplantation assayed at 2 months after transplantation. *Significantly different from lanes 1 and 2. Student t test, P < .05.

Figure 3

Culture of human cord blood CD133⁺ cells in the presence of IGFBP2 or Angptl5 stimulates ex vivo expansion of SRCs. (A) Culture of 10⁴ cryopreserved human cord blood CD133⁺ cells was initiated in serum-free StemSpan medium supplemented with 50 ng/mL human SCF, 10 ng/mL human TPO, and 50 ng/mL human Flt3-L, together with 100 ng/mL human IGFBP2 or 500 ng/mL human Angptl5. Total cell numbers were counted during the 10 days of culture period. (B) Multilineage engraftment in NOD/SCID recipients that received a transplant of cultured progenies from 8000 initial CD133⁺ cells (n = 6-9). Lanes 1-4 show cells cultured in SCF, TPO, and Flt3-L; lanes 5-8, cells cultured in SCF, TPO, Flt3-L, and IGFBP2; lanes 9-12, cells cultured in SCF, TPO, Flt3-L, and Angptl5. *Value is significantly different from the value of the lane 1 cells. Student t test, P < .05.

Figure 4

Culture of human cord blood CD133⁺ cells in the presence of Angptl5 and IGFBP2 stimulates ex vivo expansion of SRCs. (A) Culture of 10⁴ cryopreserved human cord blood CD133⁺ cells was initiated in serum-free STF medium (containing SCF, TPO, and FGF-1), or in STF medium supplemented with 500 ng/mL human Angptl5 and 500 ng/mL human IGFBP2, at 5% O₂. Total cell numbers were counted during the 11 days of culture period. (B) Extent of human chimerism in the bone marrow of NOD/SCID mice that received a transplant of 8000 or 15 000 uncultured human cord blood CD133⁺ cells, or the progenies of 8000 initial CD133⁺ cells cultured in STF medium with or without Angptl5 and IGFBP2 for 11 days. Each symbol represents the engraftment of a single mouse that underwent transplantation assayed at 2 months after transplantation (n = 7-8). *Significantly different from lanes 1-3. Student t test, P < .05. (C) Representative fluorescence-activated cell sorting (FACS) plots of bone marrow cells from one mouse at the condition represented by lane 1 of panel B (after thaw), or at the condition represented by lane 4 of panel B (cultured in STF medium containing IGFBP2 and Angptl5) at 2 months after transplantation. Percentages of cells in each quadrant are listed. (D) Summary of multilineage reconstitution from mice in lanes 2 and 4 of panel B (n = 8, respectively). Some mice that received a transplant of uncultured cells had 0% donor repopulation and these data points are not plotted. *Values are significantly different from values of the uncultured cells. Student t test, P < .05. (E) Bone marrow cells collected from mice represented by lane 4 of panel B were transplanted into secondary recipients; bone marrow aspirate from one hind leg from a primary recipient was transplanted into 2 secondary recipients. Multilineage engraftment in secondary NOD/SCID recipients was assayed at 5 to 8 weeks after transplantation (n = 12 mice underwent transplantation).

Figure 5

Limiting-dilution analysis of human cord blood CD133⁺ cells transplanted into NOD/SCID mice after culture at normal or low oxygen levels. (A,B) Culture of 2 × 10⁵ cryopreserved human cord blood CD133⁺ cells was initiated in serum-free STF medium supplemented with 500 ng/mL human Angptl5 and human 100 ng/mL IGFBP2. The numbers of total cells (A) and CD34⁺ cells (B) were counted. (C) Amount of human chimerism in the bone marrow of NOD/SCID mice that received a transplant of the indicated numbers (5000, 10 000, 20 000) of postthaw uncultured human cord blood CD133⁺ cells, or the progenies of 1667, 5000, or 10 000 CD133⁺ cells cultured in STF medium with Angptl5 and IGFBP2 in ambient oxygen (lanes 4-6) or 5% oxygen (lanes 7-9) for 10 days. Each symbol represents the engraftment of a single mouse that underwent transplantation assayed at 2 months after transplantation. Horizontal dotted lines represent arbitrary cutoffs of 0.1% and 1% reconstitution. (D,E) Limiting-dilution analysis of the repopulating ability of cells before culture (D) and after culture for 10 days in serum-free STF medium containing 500 ng/mL Angptl5 and 100 ng/mL IGFBP2 at normal or low O₂ levels (E). Plotted is the percentage of recipient mice containing less than 1% human hematopoietic populations in recipient mouse bone marrow 6 to 8 weeks after transplantation versus the number of input or input-equivalent cells injected. The progenies of 10 000 input cells cultured at normal or low O₂ repopulated all recipients and these data points (0% negative mice) are not plotted. (F) Multilineage engraftment in NOD/SCID recipients that received a transplant of 20 000 postthaw uncultured CD133⁺ cells (n = 8) or cultured progenies of 5000 initial CD133⁺ cells at normal O₂ (n = 10). Some mice that received a transplant of uncultured cells had 0% donor repopulation and these data points are not plotted.