scholarly journals Suppression of B-Cell Activation by Human Cord Blood-Derived Stem Cells (CB-SC) through the Galectin-9-Dependent Cell Contact Mechanism

2021 ◽  
Author(s):  
Wei Hu ◽  
Xiang Song ◽  
Haibo Yu ◽  
Sophia Fan ◽  
Andrew Shi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
B Cells ◽  
Cord Blood ◽  
Molecular Mechanisms ◽  
Cell Contact ◽  
Human Cord Blood ◽  
Contact Mechanism ◽  
Activated B Cells

Background: We developed the Stem Cell Educator therapy among multiple clinical trials based on the immune modulations of multipotent cord blood-derived stem cells (CB-SC) on different compartments of immune cells such as T cells and monocytes/macrophages in diabetes and other autoimmune diseases. However, the effects of CB-SC on the B cells remained unclear. To better understand the molecular mechanisms underlying the immune education of CB-SC, we explored the modulations of CB-SC on human B cells. Methods: CB-SC were isolated from human cord blood units and confirmed by flow cytometry with different markers for their purity. B cells were purified by using anti-CD19 immunomagnetic beads from human peripheral blood mononuclear cells (PBMC). Next, the activated B cells were treated in the presence or absence of coculture with CB-SC for 7 days before undergoing flow cytometry analysis of phenotypic change with different markers. RT-PCR was utilized to evaluate the levels of galectin expressions with or without treatment of activated B cells in order to find the key galectin contributing to the B-cell modulation. Results: Flow cytometry demonstrated that the proliferation of activated B cells was markedly suppressed in the presence of CB-SC, leading to the down-regulation of immunoglobulin productions from the activated B cells. Phenotypic analysis revealed that treatment with CB-SC increased the percentage of IgD+CD27- naive B cells, but decreased the percentage of IgD-CD27+ switched B cells. Transwell assay showed that the immune suppression of CB-SC on B cells was dependent on the manner of cell-cell contact via Gal-9 molecule, as confirmed by the blocking experiment with the anti-Gal-9 monoclonal antibody. Mechanistic studies demonstrated that both calcium levels of cytoplasm and mitochondria were down-regulated after the treatment with CB-SC, causing the decline of mitochondrial membrane potential in the activated B cells. Western blot exhibited that the levels of phosphorylated Akt and Erk1/2 signaling proteins in the activated B cells were also markedly reduced in the presence of CB-SC. Conclusions: CB-SC displayed multiple immune modulations on B cells through the Gal-9-mediated cell-cell contact mechanism and calcium flux/Akt/Erk1/2 signaling pathways. The data advances current understanding about the molecular mechanisms underlying the Stem Cell Educator therapy to treat autoimmune diseases in clinics.

The Polycomb Gene BMI1 Collaborates with BCR-ABL in Leukemic Transformation of Human Cord Blood CD34+ Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1350-1350
Author(s):  
Aleksandra Rizo ◽  
Sandra Olthof ◽  
OS van Ronald ◽  
Bert HJ Dontje ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
B Cells ◽  
Cord Blood ◽  
Bone Marrow Microenvironment ◽  
Leukemic Transformation ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Cd34 Cells

Abstract Previously, we demonstrated that BMI1 acts as a stem cell maintenance factor for human stem/progenitor cells. Here, we report that BMI1 collaborates with BCR-ABL in inducing leukemogenic transformation of human cord blood (CB) CD34+ cells. BMI1 and BCR-ABL were co-expressed into CB CD34+ cells (further referred as B/B cells) using a retroviral approach and cells were transplanted into NOD-SCID mice. In two out of five mice we observed leukemia within 4 months after transplantation. Chimerism levels reached 80–90% in the bone marrow and peripheral blood and morphological analysis revealed the appearance of primitive blast-like human hematopoietic cells with features that recapitulate human lymphoid leukemia. The mice were lethargic, with splenomegaly and infiltration of leukemic cells in the spleen, liver and the bone marrow and immunophenotypical analyses revealed that the cells expressed CD34 and CD19. To further understand the mechanisms underlying the leukemic transformation we performed ex-vivo long-term cultures on bone marrow stroma. We observed that the double transduced B/B cells had a strong proliferative advantage and elevated self-renewal potential as compared to controls. Expanding cultures could be maintained for over 20 weeks and Cobblestone Area Forming Cells (CAFCs) could be harvested and replated to initiate new expanding cocultures. Stem cell frequencies were determined in Long-Term Culture-Initiating Cell (LTC-IC) assays and frequencies were enhanced over 100-fold as compared to controls. Depending on the MS5 co-culture conditions, both myeloid as well as lymphoid long-term cultures could be established, indicating that extrinsic factors might dictate the lineage fate of transformed cells. To determine the necessity of a bone marrow microenvironment, we performed stroma-free liquid cultures and observed that the B/B cells were capable of expanding over 23 weeks, BMI1 cells were able to grow for 16 weeks and, importantly, BCR-ABL cells were not able to propagate long-term in stromain-dependent cultures. Thus, these data suggest that BCR-ABL cells are still dependent on cues from the bone marrow microenvironment for long-term self-renewal, and that co-expression of the intrinsic stem cell regulator BMI1 might alleviate this necessity of BCR-ABL+ cells for a microenvironment. Experiments in which B/B-transduced cells were sorted into HSC, CMP, GMP and MEP populations indicated that long-term self-renewal and expansion could particularly be imposed on the HSC population, and much less efficiently on progenitor subpopulations. In order to study whether the B/B-leukemic stem cells could be targeted by Imatinib, we applied a short pulse of Imatinib to expanding MS5 cocultures for 7 days. While the vast majority of cells in all cultures did not survive, in the B/B-transduced group a population of immature cells remained that was capable of re-initiating proliferative cultures of self-renewing CAFCs with very high frequencies (1/96 as determined by LTC-IC assays). Finally, we asked whether retroviral introduction of BMI1 in BCR-ABL+ CD34+ cells isolated from CML patients in chronic phase that expressed low endogenous BMI1 levels would affect long-term growth and self-renewal. Upon overexpression of BMI1 we observed increased proliferation capacity of the BMI1 transduced CML cells, and cultures could be maintained for much longer periods than control-transduced cultures. In conclusion, our data indicate that BMI1 collaborates with BCR-ABL in leukemic transformation, and our human-based system should provide a useful model to study the pathology of leukemias and test new drug entities.

Osteoblast-Stem Cell Contact Is Required for B-Lymphopoietic Proliferation, Not Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3635-3635
Author(s):  
Asia Gobourne ◽  
Heidi Bretscher ◽  
Alevtina Domashenko ◽  
Gerd Bungartz ◽  
Russell Garrett ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
B Cells ◽  
Cell Surface ◽  
Stromal Cells ◽  
Direct Contact ◽  
Stromal Cell ◽  
Cell Contact ◽  
B Lymphopoiesis ◽  
B Lineage

Abstract Abstract 3635 Poster Board III-571 Nonparenchymal stromal cells form the cellular constituents of tissue-specific stem cell niches. One key question in stem cell biology is the nature of signals provided by stromal cells in general, and via stem cell-stromal cell contact in particular, that direct the survival, proliferation and differentiation of tissue stem cells. We and others have previously reported that osteoblasts dictate the differentiation of hematopoietic stem cells (HSC) to B lymphopoiesis, supporting the acquisition of E2A, EBF1 and Pax5, as well as cell surface B220, CD18 and sIgM, with Flt3-ligand (FL) and interleukin 7 (IL-7) required for these activities. In this context, a fundamental unanswered question is whether, and if so why, B lymphopoiesis requires direct cell-cell contact between HSCs and osteoblasts. In the present study, we asked whether HSC contact with osteoblasts plays its principle role in B lymphopoiesis by supporting B differentiation, by supported proliferation, or both. Magnetically purified mouse Lin- Kit+ (LK) bone marrow cells were cultured for 11 days in the presence of 50 ng/ml FL plus 100 ng/ml IL-7, in direct contact with OP-9 or MS-5 osteoblastoid cells, or in proximity to OP-9/MS-5 cells but separated by a Transwell filter. Following co-culture, the hematopoietic cells were counted, the presence of B lineage-specific cell surface markers measured by flow cytometry, and the expression levels of B-lineage transcription factors E2A, EBF and Pax5 measured by quantitative PCR. LK cultured in FL + IL-7 acquired B220 as well as low levels of E2A expression, but did not express Pax5 expression nor acquire cell surface CD19 or IgM, suggesting differentiation to Pre-Pro B cells but not beyond; in contrast, LK cultured in direct contact with OP-9 differentiated fully to mature B cells, expressing Pax5 and cell surface CD19 and IgM. LK cultured above OP9 cells but with cell-cell contact prevented by an interposed Transwell also fully acquired the transcription factor and cell surface antigenic profiles of mature B cells, but the proliferation that accompanies OB-contact driven differentiation was blocked: Cells/mL Day 0 Cells/mL Day 11 Percent of Input OP-9 Direct Contact 2.3 × 105 7.8 × 106 3400% MS-5 Direct Contact 2.3 × 105 1.6 × 106 670% OP-9 Transwell 2.3 × 105 1.3 × 104 5% MS-5 Transwell 2.3 × 105 2.0 × 104 9% These data indicate that all the signals required to induce HSC differentiation to mature B cells are contained within the combination of cytokines and other molecules secreted by OP-9 cells, but that direct contact with OP-9 cells provides a proliferative stimulus not achieved by OP-9 secreted molecules, either via a direct receptor-ligand interaction or indirectly, by contact-induced secretion of specific soluble proliferative factors from osteoblasts. Experiments to distinguish these alternatives, and to identify whether such signals trigger nutrient influx, direct activation of Akt, MAPK or other key proliferative pathways, are now in progress. The proliferative role of stromal cell-parenchymal cell contact within the stem cell niche likely may have general implications to normal and neoplastic cell physiology. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Cord Blood-Derived CD133+/C-Kit+/Lin− Cells Have Bipotential Ability to Differentiate into Mesenchymal Stem Cells and Outgrowth Endothelial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Carlos Cardenas ◽  
Ja-Young Kwon ◽  
Yong-Sun Maeng
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Conditioned Medium ◽  
Mononuclear Cells ◽  
Cell Contact ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Human Cord Blood

Recent evidence suggests that mononuclear cells (MNCs) derived from bone marrow and cord blood can differentiate into mesenchymal stem cells (MSCs) or outgrowth endothelial cells (OECs). However, controversy exists as to whether MNCs have the pluripotent capacity to differentiate into MSCs or OECs or are a mixture of cell lineage-determined progenitors of MSCs or OECs. Here, using CD133+/C-kit+/Lin− mononuclear cells (CKL− cells) isolated from human umbilical cord blood using magnetic cell sorting, we characterized the potency of MNC differentiation. We first found that CKL− cells cultured with conditioned medium of OECs or MSCs differentiated into OECs or MSCs and this differentiation was also induced by cell-to-cell contact. When we cultured single CKL− cells on OEC- or MSC-conditioned medium, the cells differentiated morphologically and genetically into OEC- or MSC-like cells, respectively. Moreover, we confirmed that OECs or MSCs differentiated from CKL− cells had the ability to form capillary-like structures in Matrigel and differentiate into osteoblasts, chondrocytes, and adipocytes. Finally, using microarray analysis, we identified specific factors of OECs or MSCs that could potentially be involved in the differentiation fate of CKL− cells. Together, these results suggest that cord blood-derived CKL− cells possess at least bipotential differentiation capacity toward MSCs or OECs.

scholarly journals Co-administration of human MSC overexpressing HIF-1α increases human CD34+ cell engraftment in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Silvia Preciado ◽  
Mª Salomé Sirerol-Piquer ◽  
Sandra Muntión ◽  
Lika Osugui ◽  
Gerardo J. Martí-Chillón ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Transplantation ◽  
Attractive Potential ◽  
Human Cord Blood ◽  
Human Cd34 ◽  
Cell Engraftment

Abstract Background Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. Methods Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. Results We did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). Conclusions Co-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.

scholarly journals In Vitro Hematopoietic Reprogramming and Generation of Hematopoietic Stem Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-44-SCI-44
Author(s):  
Kateri A. Moore
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Molecular Mechanisms ◽  
Human Fibroblasts ◽  
Patient Specific ◽  
Binding Motif ◽  
Hematopoietic Stem ◽  
Mouse Fibroblasts

Hematopoietic stem cell (HSC) transplantation is widely used to treat a variety of disorders. Despite advances in the use of umbilical cord blood and mobilized stem cells, donor material remains limited. This is due to insufficient numbers of stem cells in cord blood, poor mobilization, and the lack of ethnic diversity to provide sufficient genetically matched material. Despite intensive efforts there has been limited success in generating transplantable HSCs from pluripotent stem cells (PSCs). Clearly, alternative approaches are necessary. Directly programmed hematopoietic stem/progenitor cells would provide an unlimited patient-specific source for cell replacement and genetic correction therapies as well as a platform for the future generation of patient specific therapeutics and blood products. In 2013 we demonstrated direct reprogramming of mouse fibroblasts into clonogenic hematopoietic progenitors with just four transcription factors (TFs), Gata2, Gfi1b, cFos and Etv6 (Pereira et al 2013). These four TFs induce a dynamic, multi-stage hemogenic process that progresses through an endothelial-like intermediate. As such, it appears to recapitulate definitive developmental hematopoiesis in vitro. We now have strong evidence that a similar hemogenic process can be optimally induced in human fibroblasts with Gata2, Gfi1b, and cFos. These reprogrammed cells are able to multi-lineage repopulate NSG mice. Therefore we maintain that in vitro reprogramming provides a tractable system to address the underlying molecular mechanisms of hemogenesis not possible in primary cells. We have now studied how the TFs bind DNA and initiate a molecular program that changes the epigenetic landscape to allow a change in cell fate. The studies to date have revealed cooperative binding of two of the factors to the binding motif of the other causing simultaneous silencing of fibroblast genes and activation of endothelial and hematopoietic genes. Pereira CF, Chang B, Qiu J, Niu X, Papatsenko D, Hendry CE, Clark NR, Nomura-Kitabayashi A, Kovacic JC, Ma'ayan A, Schaniel C, Lemischka IR, Moore K. Induction of a Hemogenic Program in Mouse Fibroblasts. Cell Stem Cell. 2013;13:205-218. Disclosures No relevant conflicts of interest to declare.

Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1332-1332
Author(s):  
Takahiro Suzuki ◽  
Yasuhisa Yokoyama ◽  
Keiki Kumano ◽  
Minoko Takanashi ◽  
Shiro Kozuma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Chimeric Protein ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Clinical Transplantation

Abstract [Background and purposes] Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. The use of Notch ligands or soluble IL-6 receptor combined with IL-6 has been a major technique that revealed several fold expansion of human cord blood SCID repopulating cells (SRCs). These studies, however, have been conducted in an independent manner, which hampered direct comparison and evaluation of the effect of combination of these methods. Our purpose of this study is to clarify these issues in a chemically defined serum-free medium that allows us to develop clinical usage of the culture condition. We also compared the efficiencies of SRC isolation with magnetic beads targeting CD34 and CD133. [Methods] Human cord blood CD133-sorted cells were cultured on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, flt-3 ligand, IL-3 and IL-6/soluble IL-6 receptor chimeric protein (FP6) for three weeks, and cultured cells were transplanted into NOD/SCID mice after limiting dilution to calculate the number of SRCs. To confirm whether full multipotency and self-renewal capacity of SRCs were maintained during the culture, cells were transplanted serially into NOD/SCID/γcnull (NOG) mice, and hematopoietic reconstitution was examined. To compare the efficiencies of CD34- and CD133-sorting, we divided each sample into two aliquots and separated CD34+ and CD133+ cells, and calculated the SRC numbers recovered by both separation methods. [Results and discussion] The frequencies of SRCs in the culture-initiating CD133-sorted cells and cultured progeny were calculated as one out of 1,020 and one out of 175 (adjusted to culture-initiating cells), respectively, indicating 6-fold expansion of SRCs that was statistically significant. Delta1 significantly enhanced the expansion rate of SRCs, and addition of IL-3 to this condition further promoted the expansion. In the serial transplantation assays, we found human myeloid and lymphoid reconstitution both in the primary and secondary NOG recipients, verifying the SRC capacity in the cultured cells. Notably, the CD133-sorting was approximately 4.5 times more efficient in collecting SRCs than the CD34-sorting from the same number of mononuclear cells (MNCs) (308 and 254 SRCs by CD133-sorting vs. 67 and 59 SRCs by CD34-sorting from 108 MNCs). Our study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

Suppression of IL-10 production by activated B cells via a cell contact-dependent cyclooxygenase-2 pathway upregulated in IFN-γ-treated mesenchymal stem cells

Immunobiology ◽  
2016 ◽  
Vol 221 (2) ◽  
pp. 129-136 ◽  
Author(s):  
Barbora Hermankova ◽  
Alena Zajicova ◽  
Eliska Javorkova ◽  
Milada Chudickova ◽  
Peter Trosan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
B Cells ◽  
Cyclooxygenase 2 ◽  
Cell Contact ◽  
A Cell ◽  
Ifn Γ ◽  
Activated B Cells
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