scholarly journals Red blood cells and their releasates compromise bone marrow-derived human mesenchymal stem/stromal cell survival in vitro

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ryan Christopher Dregalla ◽  
Jessica Ann Herrera ◽  
Edward Jeffery Donner
Keyword(s):  
Bone Marrow ◽  
Stromal Cell ◽  
Time Course ◽  
Bone Marrow Aspirate ◽  
Relative Number ◽  
Human Mscs ◽  
Early Passage ◽  
Cell Counts ◽  
Multiple Comparison Test

Abstract Purpose The use of bone marrow aspirate (BMA) and bone marrow aspirate concentrate (BMC) in the treatment of inflammatory orthopedic conditions has become a common practice. The therapeutic effect of BMA/BMC is thought to revolve primarily around the mesenchymal stem/stromal cell (MSC) population residing within the nucleated cell fraction. MSCs have the unique ability to respond to site of injury via the secretion of immunomodulating factors, resolving inflammation in diseased joints. Recently, the importance of hematocrit (HCT) in BMC has been debated, as the potential impact on MSC function is unknown. In the present study, we investigate MSC health over a short time-course following exposure to a range of HCT and red blood cell releasate (RBCrel) conditions. Methods Bone marrow-derived human MSCs in early passage were grown under conditions of 0%, 2.5%, 5%, 10%, 20% and 40% HCT and RBCrel conditions for 3 days. At each day, the percentage of viable, apoptotic and necrotic MSCs was determined via flow cytometry. Relative viable MSC counts in each condition was determined to account for dynamic changes in overall MSC densities over the time-course. Statistical analysis was performed using a one-way ANOVA comparing test conditions to the control followed by a Dunnett’s multiple comparison test. Results Significant reductions in viable MSCs concurrent with an increase in necrotic MSCs in high HCT and RBCrel conditions was observed within 24 h. At each successive timepoint, the percent and relative number of viable MSCs were reduced, becoming significant in multiple HCT and RBCrel conditions by Day 3. Necrosis appears to be the initial mode of MSC death following exposure to HCT and RBCrel, followed by apoptosis in surviving MSC fractions. Conclusion Various levels of HCT and RBCrel severely compromise MSC health within 3 days and HCT should be controlled in the preparation of BMC products. Further, HCT of BMCs should be routinely recorded and tracked with patient outcomes along with routine metrics (e.g. nucleated cell counts, fibroblast-colony forming units). Differences in HCT may account for the inconsistencies in the efficacy of BMC reported when treating orthopedic conditions.

scholarly journals A Method for Measurement of Drug Sensitivity of Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

2013 ◽  
Vol 18 (6) ◽  
pp. 637-646 ◽  
Author(s):  
Kristine Misund ◽  
Katarzyna A. Baranowska ◽  
Toril Holien ◽  
Christoph Rampa ◽  
Dionne C. G. Klein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Survival ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Large Scale ◽  
Stromal Cell ◽  
Marrow Stromal Cells ◽  
Cell Nuclei ◽  
Myeloma Cells

The tumor microenvironment can profoundly affect tumor cell survival as well as alter antitumor drug activity. However, conventional anticancer drug screening typically is performed in the absence of stromal cells. Here, we analyzed survival of myeloma cells co-cultured with bone marrow stromal cells (BMSC) using an automated fluorescence microscope platform, ScanR. By staining the cell nuclei with DRAQ5, we could distinguish between BMSC and myeloma cells, based on their staining intensity and nuclear shape. Using the apoptotic marker YO-PRO-1, the effects of drug treatment on the viability of the myeloma cells in the presence of stromal cells could be measured. The method does not require cell staining before incubation with drugs, and less than 5000 cells are required per condition. The method can be used for large-scale screening of anticancer drugs on primary myeloma cells. This study shows the importance of stromal cell support for primary myeloma cell survival in vitro, as half of the cell samples had a marked increase in their viability when cultured in the presence of BMSC. Stromal cell–induced protection against common myeloma drugs is also observed with this method.

Evaluation of Congenital Neutropenic Disorders by in Vitro Bone Marrow Culture

PEDIATRICS ◽  
1977 ◽  
Vol 59 (5) ◽  
pp. 739-748
Author(s):  
Peter M. Falk ◽  
Kenneth Rich ◽  
Stephen Feig ◽  
E. Richard Stiehm ◽  
David W. Golde ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Severe Disease ◽  
Stem Cell Differentiation ◽  
Peripheral Blood Cell ◽  
Clinical Expression ◽  
Essentially Normal ◽  
Cell Counts

The congenital neutropenias are a heterogeneous group of diseases whose etiology and pathogenesis are largely unknown. We studied nine neutropenic patients from seven families. Evaluation included peripheral blood cell and differential cell counts, epinephrine and typhoid vaccine stimulation studies, Rebuck skin windows, and bone marrow aspirations for morphological assessment and for in vitro culture in liquid suspension and in agar plates. Parallel cultures were set up with and without colony-stimulating activity (CSA), and peripheral leukocytes were assayed for cellular production of CSA. Patients were initially classified on the basis of their clinical course: benign, mild, moderately severe, or severe disease. One patient in the moderately severe group had an immunoglobulin disorder. Morphologically normal mature granulocytes were seen in bone marrow aspirates of two patients, and maturational defects of varying degree were seen in the remaining seven. Colony formation in agar was markedly reduced below normal in three of seven, moderately reduced in two of seven, and greater than normal in two patients. Colonies in six of seven patients consisted exclusively of macrophages. Marrow from all but one of the nine patients demonstrated poor neutrophil development in suspension culture, and addition of CSA did not result in augmented granulocytic proliferation or maturation. A scheme of normal neutrophil maturation is proposed, and the nine patients were categorized according to this scheme. Four patterns of congenital neutropenia emerged: type 1 was the most benign form of disease with essentially normal clinical and in vitro parameters, and a defect considered to be due to a small committed stem cell pool, abnormal release, or excessive utilization peripherally; type 2 had mild disease with presumed defective committed stem cell differentiation along the granulocyte line; type 3 included benign to severe clinical expression with an apparent defect at the level of the committed granulocyte precursor more severe than in type 2; type 4 disease had varied clinical expression but evidence for a defect at the level of the pluripotent stem cell.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Proteoglycan synthesis in two murine bone marrow stromal cell lines

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1777-1783 ◽  
Author(s):  
SL Kirby ◽  
SA Bentley
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Dermatan Sulfate ◽  
Cell Layer ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Proteoglycan Synthesis ◽  
Murine Bone Marrow

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3–2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3–2A does not. Cells were labeled with 35S- sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3–2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3–2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

scholarly journals Osteogenic preconditioning in perfusion bioreactors improves vascularization and bone formation by human bone marrow aspirates

2020 ◽  
Vol 6 (7) ◽  
pp. eaay2387 ◽  
Author(s):  
J. N. Harvestine ◽  
T. Gonzalez-Fernandez ◽  
A. Sebastian ◽  
N. R. Hum ◽  
D. C. Genetos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Stromal Cells ◽  
Bone Marrow Aspirate ◽  
De Novo ◽  
Trophic Factor ◽  
Calvarial Defect ◽  
Factor Secretion

Cell-derived extracellular matrix (ECM) provides a niche to promote osteogenic differentiation, cell adhesion, survival, and trophic factor secretion. To determine whether osteogenic preconditioning would improve the bone-forming potential of unfractionated bone marrow aspirate (BMA), we perfused cells on ECM-coated scaffolds to generate naïve and preconditioned constructs, respectively. The composition of cells selected from BMA was distinct on each scaffold. Naïve constructs exhibited robust proangiogenic potential in vitro, while preconditioned scaffolds contained more mesenchymal stem/stromal cells (MSCs) and endothelial cells (ECs) and exhibited an osteogenic phenotype. Upon implantation into an orthotopic calvarial defect, BMA-derived ECs were present in vessels in preconditioned implants, resulting in robust perfusion and greater vessel density over the first 14 days compared to naïve implants. After 10 weeks, human ECs and differentiated MSCs were detected in de novo tissues derived from naïve and preconditioned scaffolds. These results demonstrate that bioreactor-based preconditioning augments the bone-forming potential of BMA.

scholarly journals In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Hong Kiat Lim ◽  
Pravin Periasamy ◽  
Helen C. O’Neill
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Model Systems ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Stromal Cell Line ◽  
Reticular Cells

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

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