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Cyclic ADP-ribose generation by CD38 improves human hemopoietic stem cell engraftment into NOD/SCID mice¹

MARINA PODESTÀ^*2, ANNA PITTO^*, OSVALDO FIGARI^*, ANDREA BACIGALUPO^*, SANTINA BRUZZONE^,, LUCREZIA GUIDA^,, LUISA FRANCO, ANTONIO DE FLORA^, and ELENA ZOCCHI^,

^* Department of Hematology, 2nd Division, S. Martino Hospital, Genoa, Italy;
Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Italy;
Center of Excellence for Biomedical Research, University of Genoa, Italy; and
G. Gaslini Institute, Genoa, Italy

²Correspondence: E-mail: marina.podesta{at}hsanmartino.liguria.it

SPECIFIC AIMS

Cyclic ADP-ribose (cADPR), a potent and universal intracellular calcium mobilizer, has recently been shown to behave as a new hemopoietic cytokine as it stimulates in vitro the proliferation of human hemopoietic progenitors (HHP), both mature-committed (colony-forming cells, CFC) and immature-uncommitted (long-term culture initiating cells, LTC-IC). Therefore, we investigated in vivo the effect of cADPR on the self-renewal and expansion of hemopoietic stem cells (HSC), i.e., the most immature HHP responsible for repopulation of the bone marrow (BM), by analyzing their capacity of engraftment into irradiated NOD/SCID mice.

PRINCIPAL FINDINGS

1. In vitro priming with cADPR increases short-term HHP engraftment into NOD/SCID mice
Cord blood-derived human mononuclear cells (MNC) were first incubated for 24 h with either 100 µM ADPR (group 1, controls) or 100 µM cADPR (group 2), then infused (11.8x10⁶ MNC) into irradiated NOD/SCID mice (4 animals for each point, n=6). Mice were killed at days +20, +60, and +90 from transplant and human engraftment (CD45⁺ cells) was estimated by immunofluorescence on total BM and spleen, using anti-CD33 and anti-CD34 antibodies to identify the myeloid lineage and the progenitor cell compartments, respectively. At day +20, human engraftment (CD33⁺ and CD34⁺ cells) was higher in group 2 animals (injected with cADPR-primed MNC) vs. group 1 controls. At day +60, a marked decrease of CD33⁺ and CD34⁺ cells was observed in the BM, followed by a limited recovery at day +90. In the spleen, human cell engraftment was negligible from day +60 in both animal groups. At all time points post-transplant, however, an increased in vitro colony production from recovered BM cells was observed in the cADPR-treated group compared with controls. At day +90, the medians of produced colonies were 2232 (range 453-2510) for the controls and 4848 (range 944-7714; P<0.05) for the treated mice, respectively.

2. cADPR priming increases engraftment of HHP into secondary recipients
Primary recipient mice (groups 1 and 2) were killed at day +20 and MNC from their BM or spleen (15x10⁶ cells) were infused into secondary recipients. These were killed at day +60 to ascertain long-term engraftment. Group 2 mice showed a significantly higher secondary human engraftment in the BM, the myeloid (CD33⁺), and the more immature compartment (CD34⁺) compared with controls (P<0.05). Relevant cell numbers were 0.45 x 10⁶ vs. 0.21 x 10⁶ CD33⁺ cells and 0.43 x 10⁶ vs. 0.21 x 10⁶ CD34⁺ cells in group 2 and in controls, respectively (P<0.05). Moreover, cells from BM of group 2 secondary recipient mice exhibited significantly higher in vitro colony production than those from group 1 animals, the median values of total CFC output being 11600 vs. 8300, respectively (ranges 5300–30,780 and 1790–13,500; P<0.05).

3. Coinfusion of human CB-MNC with CD38⁺ murine feeder cell lines increases hemopoietic engraftment
Results obtained with the in vitro cADPR-primed HHP suggested to evaluate whether comparable effects could be obtained by in vivo generation of cADPR by CD38-transfected murine feeder cell lines (NIH-3T3 fibroblasts), whose ectocellular ADP-ribosyl cyclase activity was 0.58 ± 0.04 nmol cADPR/min/mg compared with undetectable values in the antisense-transfected CD38^- 3T3 cells.

NOD/SCID mice were infused with 15 x 10⁶ CB-MNC together with 10⁶ irradiated (1500 cGy) CD38^- (group 1) or CD38⁺ (group 2) murine 3T3 fibroblasts. Whereas at day +20, the two groups did not show significant differences in human engraftment, at day +60 group 2 mice had a significantly (P<0.05) higher CD34⁺ absolute cell number compared with controls (Table 1 A). The clonogenic capacity of BM cells was higher in group 2 animals than in controls, with the most significant difference being observed at day +60 (Table 1B) .

4. cADPR stimulates CD34⁺ expansion
The expansion factor (EF) of CD34⁺ HHP (i.e., the ratio between the absolute CD34⁺ cell number detected in the mouse and the number of injected CD34⁺ cells) was calculated for all protocols used in this study. In vitro priming with cADPR induced in the primary transplanted mice a slightly higher expansion of CD34⁺ HHP compared with controls at day +20 (36 vs. 29) and day +90 (16 vs. 13). In the secondary transplant, the EF was again higher for the cADPR-primed group than for the controls at day +20 (4.3 vs. 3.7) and day +60 (16.5 vs. 2.3; P<0.05).

The EF in the coinfusion experiments was comparable in the two animal groups (receiving either CD38^- or CD38⁺ stroma) at day +20, but was significantly higher (P<0.05) for the mice infused with the CD38⁺ stroma at day +60 (medians of 57.0 vs. 16.7, ranges of 7.3–177 and of 4.1–19.5, respectively). Therefore, exposure of HHP to cADPR, either by direct in vitro incubation or by coinfusion with a cADPR-generating stroma (CD38⁺), stimulates in vivo expansion of HSC.

CONCLUSIONS AND SIGNIFICANCE

We previously demonstrated that priming with cADPR stimulates in vitro proliferation and expansion of both committed (CFC) and early (LTC-IC) HHP. The expansion of LTC-IC by directly added and stroma-generated cADPR suggested a possible effect of cADPR itself on proliferation of the most immature HHP, i.e., the HSC capable of self-renewal and responsible for repopulation of the BM. Two different protocols were used to evaluate the effect of cADPR on NOD/SCID mice repopulating cells (SCID-RC): 1) an in vitro priming schedule of CB-MNC with cADPR (100 µM for 24 h), followed by their infusion into primary and then secondary recipient mice, and 2) the coinfusion of CB-MNC with cADPR-generating murine feeder cell lines (see Fig. 1 ).

View larger version (24K): [in this window] [in a new window]   Figure 1. Schematic diagram. Cyclic ADP-ribose expands human hemopoietic stem cells in NOD/SCID mice. Two protocols of cADPR treatment were followed: A) in vitro priming of human CB-MNC (HCB-MNC) with 100 µM cADPR, followed by infusion into irradiated mice (step 1, primary recipients) and, after 20 days, by withdrawal from BM or spleen and reinfusion into secondary recipient mice (step 2); B) coinfusion of HCB-MNC with CD38+ 3T3 murine fibroblasts into mice (step 4) and, after 60 days, withdrawal from BM and spleen. Counts of human cells (CD45+), immature (CD34+) and more mature (CD33+), and evaluation of their clonogenic capacity at steps 3 and 5 indicated a significant expansion of the immature HP (HSC) over controls. These were A) ADPR-treated HCB-MNC and B) HCB-MNC coinfused with natural CD38- 3T3 fibroblasts. Short-term engraftment was evaluated at day +20 (step 1); long-term engraftment was evaluated at day +60 in the secondary recipients (steps 1+2) and in mice coinfused with HHP and CD38-transfected stroma (step 4).

Our results demonstrate that cADPR enhances proliferation of HHP (CFC and LTC-IC) compared with controls, thereby improving their short-term engraftment in primary transplanted NOD/SCID mice. Moreover, cADPR proved to also induce a long-term expan-sion of HSC, as demonstrated by increased human engraftment of CD34⁺ cells in secondary transplanted mice and in mice coinfused with CB-MNC together with CD38⁺ murine feeder cell lines. In the latter condition, the remarkable in vivo expansion of the HHP seems to reflect a good efficiency of the long-lasting generation of nanomolar concentrations of cADPR by the CD38⁺ feeder cells in the BM microenvironment. Previous results on the in vitro expansion of HHP grown on CD38⁺ 3T3 fibroblasts in transwell cocultures strongly support this interpretation.

In conclusion, the present results extend the cADPR-sensitive HHP population to include the HSC and suggest two possible therapeutic applications for cADPR as a novel hemopoiesis-stimulating factor. In vitro priming of an aliquot of marrow graft with micromolar cADPR concentrations could induce a rapid in vivo overshoot of maturing myeloid elements. The protracted exposure to nanomolar cADPR concentrations, as produced by the coinfused CD38⁺ stroma, is expected to induce the expansion of long-term repopulating cells. In the clinical setting, a combination of the two protocols may reduce the aplasia period that immediately follows transplant and may improve long-term engraftment. Irradiated, in vitro expanded, CD38-transduced autologous mesenchymal cells could be coinfused together with the HP to provide a transient and localized production of cADPR in the BM microenvironment.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0520fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0520fje

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