In vitro and in vivo evaluation of cord blood hematopoietic stem and progenitor cells amplified with glycosaminoglycan mimetic

Abstract

BackgroundExpansion protocols aim at both increasing the number of umbilical cord blood (UCB) hematopoietic stem cells and progenitor cells (HSPCs) and reducing the period of neutropenia in UCB HSPC graft. Because glycosaminoglycans (GAGs) are known to be important components of the hematopoietic niche and to modulate growth factor effects, we explored the use of GAG mimetic OTR4131 to potentiate HSPC’s in vitro expansion and in vivo engraftment.MethodsUCB CD34+ cells were expanded with serum-free medium, SCF, TPO, FLT3-lig and G-CSF during 12 days in the absence or the presence of increasing OTR4131 concentrations (0-100 μg/mL). Proliferation ratio, cell viability and phenotype, functional assays, migration capacity and NOD-scid/γc-/- mice engraftment were assessed after expansion.ResultsAt Day 12, ratios of cell expansion were not significantly increased by OTR4131 treatment. Better total nucleated cell viability was observed with the use of 1 μg/mL GAG mimetic compared to control (89.6 % ± 3.7 % and 79.9 % ± 3.3 %, respectively). Phenotype analysis showed a decrease of monocyte lineage in the presence of OTR4131 and HSPC migration capacity was diminished when GAG mimetic was used at 10 μg/mL (10.9 % ± 4.1 % vs. 52.9 % ± 17.9 % for control). HSPC clonogenic capacities were similar whatever the culture conditions. Finally, in vivo experiments revealed that mice successfully engrafted in all conditions, even if some differences were observed during the first month. Three months after graft, bone marrow chimerism and blood subpopulations were similar in both groups.ConclusionsUCB HSPCs ex-vivo expansion in the presence of OTR4131 is a safe approach that did not modify cell function and engraftment capacities. In our experimental conditions, the use of a GAG mimetic did not, however, allow increasing cell expansion or optimizing their in vivo engraftment.

Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation

“…Cells were plated at a density of 40,000 cells/cm 2 in 96‐well plates. Cells were cultured at 37°C in a 5% CO 2 humidified atmosphere using IMDM culture medium supplemented with antibiotics (Anti‐anti, Sigma‐Aldrich), 10% FBS, human and SCF 100 ng/ml (PeproTech) G‐CSF 10 ng/ml (PeproTech) and IL‐3 20 ng/ml (Peprotech), adapted from (Donaldson et al , 2001; Faivre et al , 2016). For Figs 3–5, in addition to control non‐treated cells, cells were treated either with taxol (100 nM), blebbistatin (50 μM), nocodazole and blebbistatin (2 and 50 μM, respectively), or ciliobrevin (100 μM).…”

Microtubules deform the nucleus and force chromatin reorganization during early differentiation of human hematopoietic stem cells

“…We first determined the exact time window during which nucleus was reshaped as cells progressed through differentiation. To achieve this, isolated HSCs were grown in culture medium supplemented with SCF, G-CSF and IL3 to promote their differentiation into myeloid progenitors (Donaldson et al, 2001;Faivre et al, 2016) and fixed at various time points. ( Figure 3A).…”

“…Cells were plated at a density of 40 000 cells/cm 2 in 96-wells plates. Cells were cultured at 37°C in a 5% CO2 humidified atmosphere using IMDM culture medium supplemented with antibiotics (Anti-anti, Sigma Aldrich), 10% FBS, human and SCF 100 ng/ml (Peprotech) G-CSF 10 ng/ml (Peprotech) and IL-3 20 ng/ml (Peprotech), adapted from (Donaldson et al, 2001;Faivre et al, 2016). For Figures 3, 4 and 5, in addition to control non-treated cells, cells were treated either with taxol (100 nM), blebbistatin (50 µM), nocodazole and blebbistatin (2 and 50 µM respectively), or ciliobrevin (100 µM).…”

ReGeneraTing Agents (rgta®) technology combined with antibiotics improves outcomes for infections in the upper limb

“…18 RGTAs ® are able to bind numerous heparin-binding growth factors including FGF, 19 VEGF 12 and TGFβ, 19 or chemokines such as SDF-1 20 and, in doing so, protect them from proteolytic degradation and increase their bioavailability. 21 By re-establishing the spatiotemporal growth factor distribution, RGTAs ® may influence important processes contributing to tissue healing and regeneration such as angiogenesis, cell migration, and differentiation. 12,14,20,[22][23][24] RGTAs ® are biodegraded when internalized and catabolized through lysosomal pathways within cells like any other matrix element.…”

“…A notable feature of RGTAs ® is that they are able to protect and potentiate signaling peptides and growth factors, thereby re‐establishing the ECM communication network 18 . RGTAs ® are able to bind numerous heparin‐binding growth factors including FGF, 19 VEGF 12 and TGFβ, 19 or chemokines such as SDF‐1 20 and, in doing so, protect them from proteolytic degradation and increase their bioavailability 21 . By re‐establishing the spatiotemporal growth factor distribution, RGTAs ® may influence important processes contributing to tissue healing and regeneration such as angiogenesis, cell migration, and differentiation 12,14,20,22‐24 …”