Hematopoiesis is a complex process that ensures production of highly specialized blood cells, emerging from bone marrow (BM) every day.1 This continuous blood cell production is finely regulated by a molecular network of intrinsic and microenvironmental signals that control a rare pool of multipotent hematopoietic stem cells (HSCs), niched in the BM, which stands at the top of the hematopoietic hierarchy organization. During malignant hematological disorders, including acute leukemias (AL), oncogenic event(s) change(s) the key regulator of pathological hematopoietic cells (more or less differentiated) and give(s) them a capacity for unlimited self-renewal, by reversing normal proliferation control, blocking cell differentiation, and promoting resistance to apoptosis.2 Contrary to adult AL, 80% of pediatric AL are developed from lymphoid lineage (ALL). The uncontrolled proliferation of these malignant progenitors primarily affects B cells (80%) and supports tumor growth at the expense of normal blood cell production with an often-massive BM invasion with more than 90% of lymphoblasts.3 As a result, malignant niche outcompetes native hematopoietic stem and progenitor cells (HSPCs) niche by the mobilization of CD34+ cells, hence disrupting normal HSC and progenitor functions.4,5,6 Today, the status of residual naive HSPC populations in the BM at initial diagnosis or relapse in pediatric B-ALL is still poorly investigated. In this work we show that a residual hematopoiesis is still present in many patients, even when BM is fully invaded, and presents different properties depending on the B-ALL status (initial diagnosis or relapse), with residual hematopoiesis from relapsed patients being in a better shape than residual hematopoiesis from patients at initial diagnosis.

Next, we co-cultured these residual HSPCs during 14 days with murine MS-5 cells in myelo-lymphoid conditions as described by Hussein et al.7 Cells present in the supernatant were analyzed by flow cytometry to evaluate hematopoietic production and lympho-myeloid differentiation. Despite the fact that the number of sorted HSPCs was lower in relapse patients than patients at initial diagnosis (mean: 47 cells vs 230 cells, p: 0.01) (Fig. 1b), their hematopoietic production assessed by the ratio of the total number of live CD45+ cells obtained after 14 days of culture to the input number of sorted HSPCs was much better (mean 6.7 vs 0.6 for patients at initial diagnosis), and comparable to the hematopoietic production obtained with HSPCs sorted from control pediatric BM (mean 5.9) (p: 0.0001) (Fig. 1c). Interestingly, only 31.8% of CD45 cells were still alive after 14 days of cocultures with HSPCs from initial diagnosis, compared to 72.7 and 77.8% for HSPCs from relapsed and controls, respectively (p: 0.002) (Fig. 1d). No difference was observed between patients with good or poor prognosis factors (data not shown). Altogether, these results indicate that residual HSPCs present in the BM of relapsed patients are in better conditions than residual HSPCs in the BM of patients at initial diagnosis. Analysis of myeloid and lymphoid content did not show any significant difference between B-ALL (initial diagnosis or relapse) and Ct.

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