Summary Platelet transfusion has been widely used to prevent and treat life-threatening thrombocytopenia; however, preparation of a unit of concentrated platelet for transfusion requires at least 4–6 units of whole blood. At present, a platelet unit from a single donor can be prepared using apheresis, but lack of donors is still a major problem. Several approaches to produce platelets from other sources, such as haematopoietic stem cells and pluripotent stem cells, have been attempted but the system is extremely complicated, time-consuming and expensive. We now report a novel and simpler technology to obtain platelets using transdifferentiation of human bone marrow erythroblasts to megakaryocytes with overexpression of the FLI1 and ERG genes. The obtained transdifferentiated erythroblasts (both from CD71+ and GPA+ erythroblast subpopulations) exhibit typical features of megakaryocytes including morphology, expression of specific genes (cMPL and TUBB1) and a marker protein (CD41). They also have the ability to generate megakaryocytic CFU in culture and produce functional platelets, which aggregate with normal human platelets to form a normal-looking clot. Overexpression of FLI1 and ERG genes is sufficient to transdifferentiate erythroblasts to megakaryocytes that can produce functional platelets.
Activated T lymphocytes of a healthy individual were reprogrammed to induced pluripotent stem cells (iPSCs) using Sendai viral vectors. Two iPSC lines, MUSIi011-A and MUSIi011-B, were established and characterized for the expression of pluripotent markers. Both iPSC lines were able to differentiate into cells of three embryonic germ layers via embryoid body formation, exhibited normal karyotypes and were free of viral genome and transgenes at passage 15. These T lymphocyte-derived iPSCs (T-iPSCs) represent a useful starting cell source for developing next-generation immune cells such as chimeric antigen receptor (CAR)-engineered iPSC-derived T lymphocytes for the application in adoptive immunotherapy.
Allogeneic cell-based therapy is emerging as a promising approach in regenerative medicine. However, rejection of allograft due to mismatch of human leukocyte antigens (HLAs) remains a major concern after transplantation. Here, we generated a homozygous B2M knockout induced pluripotent stem cell (iPSC) line, lacking the expression of HLA class I (HLA-I) molecules, using a CRISPR/Cas9 system. The established iPSC line, MUSIi001-A-1, can serve as an in vitro model for studying immunological responses against allogeneic grafts and provides a prototype for "off-the-shelf" allogeneic cell products for future cell-based therapy.
Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.
We generated an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells (PBMNCs) isolated from a 1-year old female carrying compound heterozygote for KLF1 mutations (G176RfsX179 and A298P mutations). Non-integrating Sendai viral (SeV) vectors containing KOS, hc-MYC and hKLF4 were used for reprogramming. The established MUSIi008-A cell line contained the same mutations found in the patient, expressed pluripotent markers, differentiated into cells of three embryonic germ layers both in vitro and in vivo, and exhibited normal karyotype. This cell line may provide an alternative renewable source of cells for in vitro disease modeling of severe transfusion-dependent hemolytic anemia.
Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.
