Osteoblasts and Bone Marrow Mesenchymal Stromal Cells Control Hematopoietic Stem Cell Migration and Proliferation in 3D In Vitro Model

BACKGROUND: Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow-derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. METHODOLOGY/PRINCIPAL FINDINGS: A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34(+) cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34(+) cells was decreased. CONCLUSIONS/SIGNIFICANCE: Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation

Lenalidomide Maintenance and Measurable Residual Disease in a Real-World Multiple Myeloma Transplanted Population Receiving Different Treatment Strategies Guided by Access to Novel Drugs in Brazil

Despite recent advances in multiple myeloma (MM), the incorporation of novel agents and measurable residual disease (MRD) monitoring in low-income countries remains a challenge. Although lenalidomide maintenance (M-Len) after autologous stem cell transplantation (ASCT) has been associated with improved outcomes and MRD has refined the prognosis of complete response (CR) cases, until now, there have been no data on the benefits of these approaches in Latin America. Here, we evaluate the benefits of M-Len and MRD using next-generation flow cytometry (NGF-MRD) at Day + 100 post-ASCT (n = 53). After ASCT, responses were evaluated based on the International Myeloma Working Group criteria and NGF-MRD. MRD was positive in 60% of patients with a median progression-free survival (PFS) of 31 months vs. not reached (NR) for MRD-negative cases (p = 0.05). The patients who received M-Len continuously had a significantly better PFS and overall survival (OS) than those without M-Len (median PFS: NR vs. 29 months, p = 0.007), with progression in 11% vs. 54% of cases after a median follow-up of 34 months, respectively. In a multivariate analysis, MRD status and M-Len therapy emerged as independent predictors of PFS (median PFS of M-Len/MRD− vs. no M-Len/MRD+ of NR vs. 35 months, respectively; p = 0.01). In summary, M-Len was associated with improved survival outcomes in our real-world MM cohort in Brazil, with MRD emerging as a useful reproducible tool to identify patients at an earlier risk of relapse. The inequity in drug access remains a hurdle in countries with financial constraints, with a negative impact on MM survival.This work was supported by from Coordenação de Aperfeiçomento de Pessoal de Nível Superior—Brazil (CAPES) Finance code 001-8888.331795/2010-01; Programa de Oncobiologia 001/2017 and 004/2017; Centro Investigación Biomédica em Red—Cáncer (CIBERONC code CB//00400) of Instituto de Salud Carlos III, Ministry of Science and Innovation (Madrid, Spain), number CB16/12/00400; The International Myeloma Foundation-Black Swan Research Initiative (Los Angeles, CA) (Grant: LSHB-CT-2006-018708). A.B.S.S. was supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CAPES/PROEX, number: 88887.688096/2022-00. R.M.P. was supported by a grant from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/DGPU), number: 000281/2016-06 and CAPES/PROEX 641/2018, Brazil, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro of Brazil (FAPERJ), number: E01/200/537/2018. E.S.B. was supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CAPES/PROEX, number: 88887.335769/2019-00 and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), number: E-26/200.192/2020, Brazil

Interaction between IL-6 and TNF-α genotypes associated with bacteremia in multiple myeloma patients submitted to autologous stem cell transplantation (ASCT)

Stem cell transplantation affects patient׳s vulnerability to infections due to immunological changes related to chemotherapy. Multiple myeloma is characterized by susceptibility to infections, and IL-6 and TNF-α increased levels affect immune response (IR). Polymorphisms in promoter region of cytokine genes may alter expression levels and affect IR. We performed interaction analysis of IL-6 (−174G/C) and TNF-α (−308G/A) polymorphisms with infection susceptibility in 148 patients classified accordingly to infection status and found an interaction when compared groups with and without bacteremia (p=0.0380). The interaction may be more important than single effects for the IR associated with the infection susceptibility in ASCT

Epidemiology of hydrocephalus in Brazil

Objective: Describe the epidemiological profile and social-economic burden that hydrocephalus patients represent to the national public health system, using data available at the online database of the Brazilian Health Ministry (DataSUS). Methods: This is a populational study based on descriptive statistics of all clinical and surgical appointments included in the DataSUS database. Data included herein were collected between 2015 and 2021 and subdivided into three main groups, related to hydrocephalus incidence and mortality, hospitalizations, and financial costs. Results: In the study period, 3993 new cases of congenital hydrocephalus were diagnosed, with 6051 deaths overall. The mortality rate in the country was 1.5/100000 live births and the prevalence was 0.374/100000 inhabitants. The number of hospitalizations resulting from treatment procedures and complications of hydrocephalus was 137,880 and there was a reduction of up to 27.2% during the SARS-CoV-2 pandemics concerning previous years. Total costs for hydrocephalus management in the country amounted to 140,610,585.51 dollars. Conclusions: Hydrocephalus has a significant impact on public health budgets and pediatric mortality rates; however, it is probably underestimated, due to the paucity of demographic data and epidemiological studies in Latin America and, specifically, in Brazil. The dataSUS also has several limitations in accessing certain data related to hydrocephalus, making it difficult to have a more assertive understanding of the disease in Brazil. The results of this study provide important guidance for future research projects in clinical and experimental hydrocephalus and also the creation of public policies for better governance and care of hydrocephalus patients

PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair

Time-dependent migration of CD34⁺ cells into spheroids.

<p>At defined intervals, cells in the supernatant were collected and spheroids were harvested and trypsinized. (A) The proportion of CD34⁺ cells (R2) was determined by flow cytometry, and calculated as percentages of CD34⁺ cells inside the spheroids in relation to the total plated. (B) Dot plot of control spheroids without hematopoietic cells. (C) To distinguish migration from proliferation of cells inside the spheroids, hematopoietic cells were removed after 24 hours of co-culture, and the spheroids were maintained in culture for up to 48 hours. The number of CD34⁺ events inside the washed spheroids (closed symbols) was compared to the number of CD34⁺ events inside no washed spheroids (open symbols). (D) Time-dependent migration of CB CD34⁺ cells into simple non-induced (full line, dots), simple osteo-induced (dotted line, triangles), and mixed (dotted line, circles) spheroids. (E) The migratory profile of BM (closed squares) and CB (open squares) CD34⁺ cells in mixed spheroids is shown. Data are mean ± SEM.</p

Extracellular matrix distribution and cytoskeleton organization in simple non-induced and mixed spheroids.

<p>(A) Confocal microscopy of paraffin sections stained with H&E showing complex cellular interactions in the center of simple non-induced spheroids. (B–D) Picrosirius staining of simple non-induced (B) and mixed (C–D) spheroids showing, by optical (B–C) and confocal (D) microscopy, collagen fiber deposition restricted to the inner region of mixed spheroids. (E–J) Expression of ECM protein in mixed spheroids. Immunofluorescence staining (in green) for collagen I (E), laminin (F), collagen IV (G), osteopontin (H), and fibronectin (I–J). Osteo-induced BMSC were labeled with CM-DiI (red). A negative control is shown as an insert in (E). (K) Fibronectin expression in simple non-induced spheroids is shown for comparison. (L–O) Expression of α-SMA (L, N) and actin polymerization (M, O, phalloidin staining) in non-induced BMSC. Note the formation of stress fibers in monolayers (L–M) that are absent in 3D cultures (N–O). Nuclei were stained with DAPI (blue). (P–R) α-SMA expression (green) in mixed spheroids. Osteo-induced BMSC were labeled with CM-DiI (red in P, R). Numbers above scale bars represent the value (in micrometers) of each scale bar.</p

Gene expression profile of BMSC in 2D or 3D cultures.

<p>(A) A representative RT-PCR analysis is shown for osteo-induced BMSC cultured for 4 days as monolayers (2D ind) or as simple osteo-induced spheroids (3D ind), and non-induced BMSC cultured as monolayers (2D) or simple non-induced spheroids (3D). Blots correspond to the transcriptional factor RUNX2, the Notch ligands DELTA1 and JAG1 (Jagged-1), ANGPT1 (Angiopoietin-1), the inhibitor of Wnt pathway, DKK1, and SPP (Osteopontin). (B–E) Semiquantitative analysis is shown for GAPDH, CXCL12 (C), WNT5a (D), and KITLG (E). (n = 3, ± SEM).</p

Migration of CD34⁺ cells in mixed spheroids is dynamic.

<p>CB and BM CD34⁺ cells were co-cultured with simple or mixed spheroids for 24 hours. The supernatant was removed and the spheroids were washed and maintained in culture for more 48 hours. Phase contrast microscopy of CB CD34⁺ cells emigrating from mixed spheroids at 24 hours (A) and 48 hours (B). Number above scale bar represents the value (in micrometers) of both scale bars. Percentage of CB and BM CD34⁺ cells that migrated out from mixed and simple osteo-induced spheroids was determined by flow cytometry (E). Unlabeled cells were co-cultured with mixed spheroids for 24 hours and then the supernatants were removed and the spheroids were washed. CFSE labeled CD34⁺ cells were added to these spheroids and the co-cultures were maintained for an additional 48 hours. Representative FACS analysis showing CD34⁺ cells that were CFSE⁻ or CFSE⁺ in supernatants (C) and spheroids (D) after 48 hours of co-culture. (F) Histogram showing the percentage of CD34⁺ cells that were positive or negative for CFSE in the supernatant or spheroids after 24 (open bar) and 48 hours (grey bar). Data represent average percentages (± SEM) of CFSE⁺ and CFSE⁻ among CD34⁺ cells in one experiment with duplicates. Similar results were obtained in a second independent experiment.</p

CD34⁺ cells localize at the vicinity of osteo-induced BMSC in mixed spheroids.

<p>(A) Semithin section of simple non-induced spheroids after 48 hours of co-culture with CB CD34⁺ cells, showing numerous hematopoietic cells homogeneously distributed throughout the spheroids, even at the center. Methylene Blue. (B) Ultrastructure of the co-cultures, showing a CD34⁺ cell in contact with stromal cell projections. (C–D) Mixed spheroids co-cultured for 72 hours with CB CD34⁺ cells. Hematopoietic cells (arrows) were aligned at the interface of the two stromal cell layers (C, arrowhead) or at the vicinity of osteoid tissue (* in D). H–E. (E–F) Clusters of CD34⁺ cells (inserts) are seen at the vicinity of the osteo-induced BMSC after 48 hours. Immunohistochemistry. (H–J) Confocal microscopy of mixed spheroids co-cultured for 72 hours with CFSE labeled CB CD34⁺ cells (green, H, J). Osteo-induced BMSC were labeled with CM-DiI (red, I–J) and nuclei, that was not confocalized, were stained with DAPI, (blue, G, J). Note that hematopoietic cells are located in close proximity to osteo-induced CM-Dil⁺ BMSC but actually at the transitional region between the two cell populations. Numbers above scale bars represent the value (in micrometers) of each scale bar. (Bars in inserts  = 30 µm).</p