Murine Bone Marrow Cells Cultured Ex Vivo in the Presence of Multiple Cytokine Combinations Lose Radioprotective and Long-Term Engraftment Potential

Abstract

The desire to improve engraftment following transplantation of limited numbers of hematopoietic stem cells (HSC) has spurred the investigation of ex vivo stem cell expansion techniques. While surrogate outcomes, such as an increase in SCID-repopulating cells, suggest successful stem cell expansion in some studies, it is not clear that such assays predict outcomes using a more clinically relevant approach (e.g., myeloablation). We have addressed this by testing three cytokine combinations for their ability to increase the radioprotective and long-term marrow reconstitution capacity of hematopoietic cells cultured ex vivo. Low numbers of light-density (LD) mouse bone marrow (BM) cells or their expanded product were injected into lethally irradiated (9 Gy) congenic recipients. Survival rates and percent donor engraftment were compared at 2, 5, and 7 months post-transplant. The three cytokine combinations used were: (i) kit-ligand (L), thrombopoietin (Tpo), Flt-3 L; (ii) cytokines in (i) plus interleukin-11 (IL-11); (iii) cytokines in (ii) plus IL-3. At 7 months post-transplant, LD cell doses of 10(4), 2-2.5 x 10(4), and 0.5-1.0 x 10(5) gave predictable survivals of 20-30%, 40-70%, and 100%, respectively. Mean percent donor engraftments were 54.9% (SEM 36%), 55.7% (SEM 36%), and 76.3% (SEM 21%), respectively. When cells expanded for 3 or 5-7 days with the various cytokine combinations were transplanted into different groups of mice, survival rates and percent donor engraftment were almost uniformly poorer than results obtained with unmanipulated cells, and cells expanded for 5-7 days led to poorer outcomes than cells expanded for 3 days. Overall, ex vivo expansion of LD BM cells with the cytokine combinations chosen failed to improve transplant outcomes in this model.

Tumor Necrosis Factor Receptors Support Murine Hematopoietic Progenitor Function in the Early Stages of Engraftment

“…Notably, insights into the negative regulatory effect of TNF-a over colony formation [8,13,14,17,20], proapoptotic effects [18,19], and impaired hematopoietic reconstitution capacity [8,13,14,[17][18][19] have been collected using pretransplant culture of the progenitors. It is well recognized that extended culture prior to transplantation is commonly associated with impaired hematopoietic engraftment of cycling progenitors independent of the activity of TNF-a [41][42][43], and under such conditions TNF-a might appear to correlate to negative regulation [7][8][9].…”

Recombinant IL-7/HGFβ efficiently induces transplantable murine hematopoietic stem cells

“…Of the numerous cytokines that have been tested, none has thus far been able to stimulate the generation of sufficient functional HSCs for clinical application. This negative effect has been attributed to the decline or loss of long-term in vivo repopulating abilities (4,5,8) secondary to the entrance of HSCs into cell cycle (9)(10)(11). Therefore, only cytokines that maintain HSCs in G 0 phase, such as SDF-1, TGF-β, FGF, and angiopoietin-1, have been found to preserve their engraftment ability (12)(13)(14)(15).…”

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells

“…Compromised long-term repopulating activity following ex vivo expansion has been reported in fetal sheep, [18][19][20] nonhuman primate, 21 feline 22 and mouse models. [23][24][25] Clinically, while the absence of durable engraftment from ex vivo-expanded CD34 þ cells has once been reported, 26 durable engraftment has been reported in patients who received expanded autologous peripheral blood progenitor cells as the sole source of hematopoietic support following high-dose therapy. 19 This observation, together with other evidence, suggests that the primitive HPC compartment, [27][28][29][30][31][32] HPC homing after transplantation 33 and basic biological and genetic characteristics of HPC, 34 are preserved following ex vivo expansion.…”