scholarly journals Role of ex vivo Expanded Mesenchymal Stromal Cells in Determining Hematopoietic Stem Cell Transplantation Outcome

2021 ◽  
Vol 9 ◽  
Author(s):  
Stefania Crippa ◽  
Ludovica Santi ◽  
Margherita Berti ◽  
Giada De Ponti ◽  
Maria Ester Bernardo
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Late Complications ◽  
Point Of View ◽  
Human Organism ◽  
Paracrine Factors ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
The One

Overall, the human organism requires the production of ∼1 trillion new blood cells per day. Such goal is achieved via hematopoiesis occurring within the bone marrow (BM) under the tight regulation of hematopoietic stem and progenitor cell (HSPC) homeostasis made by the BM microenvironment. The BM niche is defined by the close interactions of HSPCs and non-hematopoietic cells of different origin, which control the maintenance of HSPCs and orchestrate hematopoiesis in response to the body’s requirements. The activity of the BM niche is regulated by specific signaling pathways in physiological conditions and in case of stress, including the one induced by the HSPC transplantation (HSCT) procedures. HSCT is the curative option for several hematological and non-hematological diseases, despite being associated with early and late complications, mainly due to a low level of HSPC engraftment, impaired hematopoietic recovery, immune-mediated graft rejection, and graft-versus-host disease (GvHD) in case of allogenic transplant. Mesenchymal stromal cells (MSCs) are key elements of the BM niche, regulating HSPC homeostasis by direct contact and secreting several paracrine factors. In this review, we will explore the several mechanisms through which MSCs impact on the supportive activity of the BM niche and regulate HSPC homeostasis. We will further discuss how the growing understanding of such mechanisms have impacted, under a clinical point of view, on the transplantation field. In more recent years, these results have instructed the design of clinical trials to ameliorate the outcome of HSCT, especially in the allogenic setting, and when low doses of HSPCs were available for transplantation.

scholarly journals Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells

2021 ◽  
Author(s):  
Valentina Orticelli ◽  
Andrea Papait ◽  
Elsa Vertua ◽  
Patrizia Bonassi Signoroni ◽  
Pietro Romele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells

2015 ◽  
Vol 39 (10) ◽  
pp. 1099-1110 ◽  
Author(s):  
Iordanis Pelagiadis ◽  
Eftichia Stiakaki ◽  
Christianna Choulaki ◽  
Maria Kalmanti ◽  
Helen Dimitriou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

Extracellular Vesicles Isolated from Mesenchymal Stromal Cells Primed with Hypoxia: Novel strategy in Regenerative Medicine

2020 ◽  
Vol 15 ◽  
Author(s):  
Shalmali Pendse ◽  
Vaijayanti Kale ◽  
Anuradha Vaidya
Keyword(s):  
Extracellular Vesicles ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Cell Types ◽  
Molecular Profile ◽  
Cell Contact ◽  
Hematopoietic Stem

: Mesenchymal stromal cells (MSCs) regulate other cell types through a strong paracrine component called the secretome, comprising of several bioactive entities. The composition of the MSCs’ secretome is dependent upon the microenvironment in which they thrive, and hence, it could be altered by pre-conditioning the MSCs during in vitro culture. The primary aim of this review is to discuss various strategies that are being used for pre-conditioning of MSCs, also known as “priming of MSCs”, in the context of improving their therapeutic potential. Several studies have underscored the importance of extracellular vesicles (EVs) derived from primed MSCs in improving their efficacy in the treatment of various diseases. We have previously shown that co-culturing hematopoietic stem cells (HSCs) with hypoxiaprimed MSCs improves their engraftment potential. Now the question we pose is would priming of MSCs with hypoxiafavorably alter theirsecretome and would this altered secretome work as effectively as the cell to cell contact did? Here we review the current strategies of using the secretome, specifically the EVs (microvesicles and exosomes), collected from the primed MSCs with the intention of expanding HSCs ex vivo. We speculate that an effective priming of MSCs in vitrocould modulate the molecular profile of their secretome, which could eventually be used as a cell-free biologic in clinical settings.

scholarly journals Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Olga Schmal ◽  
Jan Seifert ◽  
Tilman E. Schäffer ◽  
Christina B. Walter ◽  
Wilhelm K. Aicher ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Three Dimensional ◽  
3D Culture ◽  
Hanging Drop ◽  
Mixed Cell ◽  
Hematopoietic Stem ◽  
Extracellular Matrix Components ◽  
Stem And Progenitor Cells

Efficientex vivoexpansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

Cord Blood Hematopoietic Progenitor Cell in Co-Culture with Bone Marrow Mesenchymal Stromal Cells: A Synergic Effect

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4811-4811
Author(s):  
Camillo Almici ◽  
Arabella Neva ◽  
Rosanna Verardi ◽  
Simona Braga ◽  
Andrea Di Palma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cell ◽  
Cord Blood Sample ◽  
Hematopoietic Stem

Abstract Abstract 4811 The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone Marrow-Derived Mesenchymal Stromal Cells: A Novel Target to Optimize Hematopoietic Stem Cell Transplantation Protocols in Hematological Malignancies and Rare Genetic Disorders

2019 ◽  
Vol 9 (1) ◽  
pp. 2 ◽  
Author(s):  
Stefania Crippa ◽  
Ludovica Santi ◽  
Roberto Bosotti ◽  
Giulia Porro ◽  
Maria Ester Bernardo
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stromal Cells ◽  
Hematological Malignancies ◽  
Ex Vivo ◽  
Genetic Disorders ◽  
Hematopoietic Stem ◽  
Bm Niche

Mesenchymal stromal cells (MSCs) are crucial elements in the bone marrow (BM) niche where they provide physical support and secrete soluble factors to control and maintain hematopoietic stem progenitor cells (HSPCs). Given their role in the BM niche and HSPC support, MSCs have been employed in the clinical setting to expand ex-vivo HSPCs, as well as to facilitate HSPC engraftment in vivo. Specific alterations in the mesenchymal compartment have been described in hematological malignancies, as well as in rare genetic disorders, diseases that are amenable to allogeneic hematopoietic stem cell transplantation (HSCT), and ex-vivo HSPC-gene therapy (HSC-GT). Dissecting the in vivo function of human MSCs and studying their biological and functional properties in these diseases is a critical requirement to optimize transplantation outcomes. In this review, the role of MSCs in the orchestration of the BM niche will be revised, and alterations in the mesenchymal compartment in specific disorders will be discussed, focusing on the need to correct and restore a proper microenvironment to ameliorate transplantation procedures, and more in general disease outcomes.

scholarly journals Application of conditioned medium from mesenchymal stromal cells in the protocol for ex vivo production of megakaryocytes and platelets

2021 ◽  
Vol 66 (4) ◽  
pp. 526-538
Author(s):  
D. Yu. Klyuchnikov ◽  
M. Yu. Yazykova ◽  
A. A. Stepanov ◽  
S. E. Volchkov ◽  
O. V. Tyumina
Keyword(s):  
Cord Blood ◽  
Conditioned Medium ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Blood Platelets ◽  
Venous Blood ◽  
Hematopoietic Stem ◽  
Group 2 ◽  
Group 1

Introduction. Of interest is the use of a conditioned medium from mesenchymal stromal cells in order to increase the expansion of CD34+  hematopoietic stem cells (HSCs).Aim — to analyze the effi cacy of two methods of ex vivo production of human megakaryocytes and platelets from CD34+ cord blood HSC using conditioned media from mesenchymal stromal cells and IMDM. Methods. Two cultivation methods that differ from each other by medium composition were compared. As a control of antigen expression of the donor, venous blood platelets were used. CD34+ HSCs were isolated from mononuclear fraction of cord blood using the immunomagnetic selection technique. The resulting cells were introduced at a concentration of 1 × 104  cells/mL into 24-well plates and cultured at 39 °C and 10 % CO2  for the first 7 days, after which the conditions were changed to 37 °C and 5 % CO2  and cultured for 14 days. In Group 1, up to day 7, the culture was performed using conditioned medium from mesenchymal stromal cell containing TPO (30 ng/mL), SCF (2 ng/mL), IL-6 (7.5 ng/mL), IL-9 (13.5 ng/mL), and in Group 2 a IMDM medium with the same cytokine cocktail was used. The cells were calculated using haemocytometer. CD34, CD41a, CD42b expression was evaluated using fl ow cytometry. Statistic data was processed with using R-language. The differences were evaluated as statistically signifi cant at signifi cance level p < 0.05.Results. Megakaryocyte production was observed starting from day 7 of culture. The expression level using conditioned medium from mesenchymal stromal cells (Group 1) according to CD41a was 5.84 ± 0.33 % versus 10.43 ± 1.08 % using IMDM medium (Group 2). On day 13 the ratio increased up to 42.05 ± 1.71 % in Group 1 and 61.78 ± 1.71 % in Group 2. CD41a+ megakaryocytes of Group 1 expressed the CD42b marker at the level of 96.85 ± 1.06 % versus 88.7 ± 0.56 % in Group 2. With the application of MSC conditioned medium the average number of nucleated cells was signifi cantly higher on the day 11 and it was equal 326.016 ± 1.86 × 104  cells/mL vs 197.26 ± 10.55 × 104  cells/mL in IMDM medium. Proplatelet formation was observed with microscopy staring from the day 12. The ratio of CD41a+ /CD42b+  platelets was 59.5 ± 3.85 % in conditioned medium, 65.9 ± 8.72 % in IMDM, and 96.11 ± 0.89 % in control platelets derived from venous blood.Conclusion. It was demonstrated that the use of MSC conditioned medium leads to an increase in the expansion of nucleated cells, however it decreases the rate of differentiation in megakaryocytes. 

scholarly journals Hematopoietic Stem Cell Regeneration through Paracrine Regulation of the Wnt5a-Prox1 Signaling Axis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 201-201
Author(s):  
Qiqi Lin ◽  
Srinivas Chatla ◽  
Limei Wu ◽  
Fabliha Chowdhury ◽  
Wei Du
Keyword(s):  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Paracrine Factors ◽  
Hematopoietic Stem ◽  
Aged Mice ◽  
Hematopoietic Recovery ◽  
Bm Niche ◽  
Genes Encoding ◽  
Signaling Axis ◽  
Homeobox Transcription Factor

Abstract The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a protein, which is produced in LepR +CXCL12 + BM stromal cells. Wnt5a treatment of irradiated FA mice enhances hematopoietic recovery and HSC regeneration. Conversely, Wnt5a neutralization in co-cultured LepR + BM stromal cells inhibits HSC regeneration and hematopoietic recovery following TBI. Mechanistically, Wnt5a secreted by LepR +CXCL12 + BM stromal cells inhibits b-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSPCs. The detrimental effect of deregulated Wnt5a-Prox1 signaling on HSC regeneration and hematopoietic recovery is also observed in aged mice. Irradiation induces upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improves HSC regeneration and hematopoietic recovery after irradiation. Finally, treatment of aged mice with Wnt5a enhances hematopoietic repopulation. Collectively, these findings identify the novel paracrine Wnt5a-Prox1 signaling axis in regulating HSC regeneration under conditions of injury and aging. Disclosures No relevant conflicts of interest to declare.

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