Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells.

BACKGROUND: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. METHODS: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of HBB and HBG2. RESULTS: Both 2D and 3D differentiation protocols led to the induction of CD34+/CD43+ HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71+/CD235a+ cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a+/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. CONCLUSION: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.

Image-guided percutaneous intralesional administration of mesenchymal stromal cells in subjects with chronic complete spinal cord injury: a pilot study.

BACKGROUND AIMS: The potential of cell therapies to improve neurological function in subjects with spinal cord injury (SCI) is currently under investigation. In this context, the choice of cell type, dose, route and administration regimen are key factors. Mesenchymal stromal cells (MSCs) can be easily obtained, expanded and are suitable for autologous transplantation. Here we conducted a pilot study that evaluated safety, feasibility and potential efficacy of intralesional MSCs transplantation performed through image-guided percutaneous injection, in subjects with chronic complete SCI. METHODS: Five subjects with chronic traumatic SCI (>6 months), at thoracic level, classified as American Spinal Cord Injury Association impairment scale (AIS) grade A, complete injury, were included. Somatosensory evoked potentials (SSEP), spinal magnetic resonance imaging (MRI) and urodynamics were assessed before and after treatment. Autologous MSCs were injected directly into the lesion site through percutaneous injection guided by computerized tomography (CT). RESULTS: Tomography-guided percutaneous cell transplantation was a safe procedure without adverse effects. All subjects displayed improvements in spinal cord independence measure (SCIM) scores and functional independence measure (FIM), mainly due to improvements in bowel movements and regularity. Three subjects showed improved sensitivity to tactile stimulation. Two subjects improved AIS grade to B, incomplete injury, although this was sustained in only one of them during the study follow-up. CONCLUSION: Autologous bone marrow MSC transplantation, performed through CT-guided percutaneous injection, was shown to be safe and feasible. Further studies are required to demonstrate efficacy of this therapeutic scheme.