Mesenchymal Stromal Precursor Cells from the Bone Marrow of Acute Myeloid and Lymphoid Leukemia Patients: Characteristics in Newly Diagnosed, before and after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Hematopoiesis is maintained in close contact with bone marrow (BM) stroma. Malignant hematopoietic cells probably affect the hematopoietic microenvironment, as well as high dose chemotherapy. The goal of this study was to analyze the alterations in the characteristics of human multipotent mesenchymal stromal cells (MSC) and their more differentiated progeny – fibroblastic colony forming units (CFU-F), derived from the BM of acute myeloid and lymphoid leukemia (AML and ALL) patients. Methods 26 newly diagnosed cases (18 AML, 8 ALL) and 35 patients (20 AML, 15 ALL) before and during 1 year after allogeneic hematopoietic stem cell transplantation (alloHSCT) were involved in the study after informed consent. BM was aspirated prior to any treatment in the newly diagnosed group and before the conditioning and at 6 time points during 1st year after the alloHSCT. MSC were cultured in aMEM with 10% FCS. Cumulative MSC production was counted after 3 passages. CFU-F concentration was analyzed in BM samples by standard protocol. The relative expression level (REL) of genes was measured by RT2 Profiler PCR Array (Qiagen) and TaqMan RQ-PCR. MSC and CFU-F from 50 healthy donors of BM for alloHSCT were used as a control after informed consent. Results The CFU-F concentration in the BM of patients at the moment of diagnostics was 1/3 of the donor’s for AML and ≈1/10 of ALL (9±5.2 and 3.9±2.3 versus 31±3.5 per 106 nucleated cells in donor BM, p<0.05). Most of the patients assigned to alloHSCT were in the remission and at that moment CFU-F concentration in their BM differed insignificantly from the donors’ one. After alloHSCT CFU-F concentration in AML and ALL patients’ BM decreased 3-9 folds for the whole next year. The decrease at each time point was highly significant comparing to donors. MSC properties were also altered in patients with acute leukemia. Cumulative cell production in MSC of newly diagnosed AML and ALL patients did not differ from donors’ one. MSC production from the BM of patients after standard courses of chemotherapy (for AML patients -mainly various numbers of “7+3” blocks and for ALL patients - ALL-2009) before alloHSCT did not differ from donors, while after alloHSCT cumulative AML and ALL MSC production decreased 1.3-6.3 folds for the next year. The decrease at almost each time point was significant comparing to donors. Gene expression analysis revealed that in MSC at the moment of AML diagnosis the REL of FGF2, IL1B, IL6, JAG1, PDGFB, VCAM1, VEGFA decreased more than 2 fold. Prior to alloHSCT the REL of IL6 and IL1B were still very low, after the alloHSCT the REL of IL6, JAG1, PDGFB increased, while IL1B stayed at the low level at least for 6 months. In MSC at the moment of ALL diagnosis the REL of IFNG, IGF1, IL6, KITLG, NES, PTPRC, TBX5 decreased more than 5 fold, while these of CSF3, ICAM1, IL1B, ITGAX – increased more than 5 fold. REL of IL6 and IDO1 were elevated prior to alloHSTC and stayed at such level for a year. Prior to alloHSCT the REL of FGFR2, PDGFRA, SDF1 and FGF2 were very low, after the alloHSCT the REL of JAG1, FGF2, TGFB1 slightly increased, while FGFR2, PDGFRB, SDF1 stayed at the low level at least for 1 year. Conclusions During the leukemia development malignant cells alter the stromal precursor cells leading to the decrease in the REL of many regulatory genes and in the number of more differentiated stromal precursor cells in the BM. Chemotherapy used for induction of the remission in these patients restore functional ability of MSC and CFU-F, while gene expression levels remained altered. Conditioning regiments used for the alloHSCT significantly damage both types of studied stromal precursors, and the effect lasted at least for 1 year. So, both leukemic cells and chemotherapy affect BM hematopoietic microenvironment. Disclosures No relevant conflicts of interest to declare.

Stromal Precursor Cells Characteristics In Patients With ALL Before and After Allogeneic Hematopoietic Stem Cell Transplantation

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow (BM). Lymphocytes connect tightly with stromal cells in BM. Leukemia and high dose chemotherapy affected both hematopoietic and stromal precursor cells. Changes in the hematopoiesis that occur during acute lymphoblastic leukemia (ALL) probably correlate with variation in the composition of stromal microenvironment. The aim of the study was to analyze the alterations occurring in stromal precursor cells - colony forming unit fibroblasts (CFU-F) and multipotent mesenchymal stromal cells (MSC) in patients with ALL before and after allogeneic hematopoietic stem cell transplantation (HSCT). HSCT following myeloblative conditioning was performed in 7 ALL patients (6 male, 1 female). After informed consent BM was aspirated before conditioning, and during the year after transplantation. MSC were cultured in aMEM with 10% fetal calf serum. Cumulative MSC production was counted after 5 passages. CFU-F was analyzed in standard conditions. The relative expression level (REL) of different genes was measured by RT-PCR. All data obtained at different time points were analyzed summarily. As a control MSC and CFU-F from 20 healthy donors of BM for HSCT were used after informed consent. The concentration of CFU-F in BM of ALL patients before HSCT was reduced by 12% in comparison with donors. After HSCT the concentration of CFU-F decreased further in 5.2 fold compared to the level before HSCT (p=0.02). Colonies formed by CFU-F depend on autocrine secretion of FGF2. It signals through 2 types of receptors, FGFR1 is considered to be the most important one. REL of FGFR1 was reduced by 27% in colonies from BM of patients before HSCT compared to donors. After HSCT it further decreased 1.2 fold. REL of FGFR2 in colonies of patients decreased 16 fold before and 50 fold after HSCT compared to donors (p=0.01 in both cases). REL of FGF2 in colonies of patients before HSCT doubled compared to donors and decreased 16 fold after HSCT. These data offer the molecular basis of decrease in CFU-F concentration in BM of patients after HSCT. In CFU-F colonies from patients REL of FGFR2 was decreased dramatically, however before HSCT it did not lead to decrease in CFU-F concentration due to increase in REL of FGF2. After HSCT REL of FGF2 and both receptors considerably decreased that led to significant reduction of CFU-F concentration in patients BM. REL of genes-markers of adipogenic and osteogenic differentiations were significantly decreased in CFU-F colonies of patients before HSCT (SPP1 in 8.6 fold, p=0.02, PPARG in 4.7 fold, p=0.01) pointing to less differentiated status of CFU-F progeny. After HSCT REL of these genes increased up to levels higher (PPARG in 3.5 fold, p=0.02) or comparable with donor ones, thus CFU-F seemed to regain “normal” differentiation status. REL of chondrogenic marker SOX9 did not change in CFU-F of ALL patients before and after HSCT, probably due to block of this differentiation lineage in CFU-F. REL of BMP4 in patients’ CFU-F colonies was reduced 30 fold compared to donors (p=0.002), apparently due to damage made by leukemic сells. It increased 100 fold compared to donors after HSCT, that might reflect the intensive recovery of stromal microenvironment by interaction with non-malignant hematopoietic cells. Thus multiple alterations in CFU-F concentration and gene expression were revealed in CFU-F from BM of patients. MSC were also affected in ALL patients. Cumulative cell production in cultures from patients before HSCT was reduced by 35%, while after HSCT the decrease was more pronounced (by 61%, p=0.01). REL of genes regulating the proliferation (FGFR1, FGFR2 and FGF2) in patients MSC was halved in comparison to donors. After HSCT REL of both types of receptors did not change, while REL of FGF2 increased 1.5 fold not reaching donors’ level. The decrease in cumulative cell production could depend on both FGF2 pathway alteration and on the increase of differentiation status in MSC population after HSCT. REL of differentiation markers in MSC before HSCT did not differ from donors’ ones, while after HSCT REL of SPP1 increased 3.8 fold, PPARG – 2.3 fold (p=0.05) and SOX9 in 1.6 fold compared to donors. In patients MSC were less affected by leukemia and its treatment than CFU-F. This data suggest significant damage of stromal precursor cells in ALL patients that lasted at least for one year since HSCT. Disclosures: No relevant conflicts of interest to declare.