Stromal Precursors in the Bone Marrow of Untreated Patients with Severe and Non-Severe Aplastic Anemia Differ in the Proliferative Potential

Background. Aplastic anemia (AA) is a disorder characterized by pancytopenia, hypoplastic bone marrow (BM), and the absence of underlying malignancy. It is believed to be of autoimmune nature. However, some patients fail to respond to the immunosuppressive therapy. The impaired hematopoietic microenvironment could be another reason for BM failure. The severity of AA varies widely from mild, chronic pancytopenia to total hematopoietic failure. The diagnosis of severe (SAA) and non-severe AA (NAA) is based on an absolute neutrophil count as an essential criterion. The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in BM of untreated SAA and NAA patients. Methods. The study included 22 AA patients (8 with SAA and 14 with NAA) in the debut of the disease. In all patients BM was aspirated after informed consent at diagnostic punctures. The proportion of non-hematopoietic CD45-CD34-CD71-CD235-CD90+CD73+CD105+ cells was estimated by FACS. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. Individual CFU-F-derived colonies were analyzed for their proliferative and differentiation potential. Adipogenic and osteogenic differentiation potential was analyzed with standard techniques. Relative expression level (REL) of several genes had been estimated with RT-PCR in Taqman modification. As a control 19 BM samples of healthy donors of according age were used. Results. The data are presented in the table. The proportion of non-hematopoietic cells was higher in the BM of AA patients than in healthy donors. We recalculated the proportion of CFU-F among non- hematopoietic cells; it was similar in the BM of AA patients and healthy donors. However, the concentration of -CFU-F was much higher in the BM of patients with SAA then in the BM of patients with NAA. Among NAA patients, 2 had PNG clone and unlike other NAA patients increased CFU-F concentration, comparable to patients with SAA. It seems that the character of stromal cell damage depends on the severity of AA. Individual CFU-F- derived clones from the BM of NAA patients had very limited proliferative potential, while those of SAA patients did not differ from colonies of healthy donors. The analysis of CFU-F-derived colonies differentiation ability revealed that the proportion of the precursors that did not respond to the differentiation induction was higher in the BM of AA patients than in donors. It reflects the involvement of a certain subpopulation of stromal precursors that are either pre-differentiated into fibroblasts, or, conversely, earlier precursors of the hematopoietic microenvironment, which were not able to differentiate into osteoblasts and/or adipocytes within standard time. The analysis of the MMSC growth characteristics revealed that the time required for MMSC from SAA and NAA patients to form a confluent monolayer after the initial seeding and the population doubling time, were significantly higher than in MMSC of healthy donors. Thus, the proliferation rate of MMSC of AA patients is reduced. Nevertheless, the total cell production for 3 passages did not differ in cultures of AA patients and healthy donors. Therefore, the proliferative potential of MMSC of AA patients is not altered. Probably MMSC being analyzed ex vivo can restore their function. However, the analysis of REL of genes regulating the proliferation (FGF2, FGFR1, FGFR2) in MSCs had revealed the differences in comparison with donors and between SAA and NAA. Moreover, the analysis of the polymorphism in CFH gene, participating in immunomodulation, showed that the distribution differs between NAA and SAA patients. Conclusions. Stromal precursors in BM of untreated NAA and SAA patients are impaired and differ between the two subtypes of AA. It seems that the differences between NAA and SAA may lay not only in the absolute neutrophil count but also in the BM stroma itself. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the hematopoiesis failure. This work was supported by the Russian Foundation for Basic Research, project no. 19-015-00280. Table Disclosures No relevant conflicts of interest to declare.

Stem Cells Defects in Systemic Sclerosis.

Systemic Sclerosis (SSc) is a connective tissue disease characterized by early generalized microangiopathy and culminating in systemic fibrosis. Recent studies have provided evidence that SSc is associated with a reactive but ineffective angiogenesis, so that the disease finally leads to the irreversible loss of capillaries. Aim of the study was to investigate whether impaired vasculogenesis in SSc is due to defective characteristics in BM microenvironment. Peripheral blood (PB) samples were collected from 70 patients (pts): circulating endothelial progenitors (CEPs) were characterized as CD45−/CD133+ and evaluated by flow cytometry. BM samples were collected from 14 SSc pts and hematopoiesis evaluated by various assays. CD133+ cells were isolated by immunomagnetic sorting (IMS) and grown in order to induce endothelial differentiation. Long-term bone marrow cultures (LTBMC) were assessed and the number of stromal clonogenic precursors evaluated by a CFU-F (colony-forming unit fibroblast) assay. Mesenchymal stem cells (MSC) were separated by IMS for the expression of the nerve growth factor-receptor (NGF-R+) and grown in order to assess the clonogenic potential and the proliferative capacity, while their multipotential differentiation ability was determined after culture in different conditioned media. Phenotypic analysis of BM mononuclear cells showed a greater expression of the surface markers P1H12 and CD105 TGF-β receptor (1.2%±0.6 vs 0.5%±0.1 in normal controls, p=0.01 and 9.9%±5 vs 4.7%±3, p=0.02 respectively), but lower percentages of NGF-R+ stromal cell precursors (0.73±0.5 vs 1.61±0.6, p=0.02) and CD133+ cells (0.36%±0.4 vs 1.2%±0.8, p=0.05). On the contrary, the absolute number of CEPs in PB was higher in patients with SSc than in healthy controls (mean values 2.1 cells/μL vs 0.26 cells/μL, p=0.04). When BM CD133+ cells were grown in the presence of VEGF, only 3/12 cases gave endothelial differentiation, but always with a reduced proliferative ability. All pts showed a defective stromal compartment and a reduced number of BM stromal precursors, as detected by the LTBMC and by the lower CFU-F frequency (4%±3.2 vs 43%±19.8/1x10(e)6 LDMNCs, p=0.002 and 7±12.8 vs 69±61/1x10(e)5 NGF-R+ cells, p=0.01). Interestingly, NGF-R+ MSC overexpressed KDR and CD117 (26.4%±7.4 vs 4.6%±1.7, p=0.01 and 87.7%±5.1 vs 57.6%±11, p=0.03 respectively): when grown in the presence of VEGF they gave rise to endothelial colonies, only in 2/8 cases they formed a confluent layer with fibroblastic morphology but a reduced proliferative ability, while in the presence of adipogenic or osteogenic inductive media they failed to origin specific differentiation. Moreover, all “in vitro” differentiated endothelial cells even before activation showed high levels of CD62-E, VCAM-1 and CD105 expression, suggestive of the presence of increased levels of proangiogenic factors in BM. The results of this study provide evidence that patients with SSc have a stem cell defect involving both the hematopoietic and the stromal cells compartments. The higher expression of KDR on NGF-R+ cells suggests a role for VEGF in inducing endothelial differentiation of MSC, so resulting in a depletion of stromal precursors. The continuous recruitment of endothelial progenitors to sites of vascular injury, suggested by the high numbers of CEPs in PB, might lead to the irreversible BM damage we observed.