Recent Updates on Induced Pluripotent Stem Cells in Hematological Disorders

Over the past decade, enormous progress has been made in the field of induced pluripotent stem cells (iPSCs). Patients’ somatic cells such as skin fibroblasts or blood cells can be used to generate disease-specific pluripotent stem cells, which have unlimited proliferation and can differentiate into all cell types of the body. Human iPSCs offer great promises and opportunities for treatments of degenerative diseases and studying disease pathology and drug screening. So far, many iPSC-derived disease models have led to the discovery of novel pathological mechanisms as well as new drugs in the pipeline that have been tested in the iPSC-derived cells for efficacy and potential toxicities. Furthermore, recent advances in genome editing technology in combination with the iPSC technology have provided a versatile platform for studying stem cell biology and regenerative medicine. In this review, an overview of iPSCs, patient-specific iPSCs for disease modeling and drug screening, applications of iPSCs and genome editing technology in hematological disorders, remaining challenges, and future perspectives of iPSCs in hematological diseases will be discussed

Advances in Adoptive Cell Therapy Using Induced Pluripotent Stem Cell-Derived T Cells

Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR) T cells holds impressive clinical outcomes especially in patients who are refractory to other kinds of therapy. However, many challenges hinder its clinical applications. For example, patients who undergo chemotherapy usually have an insufficient number of autologous T cells due to lymphopenia. Long-term ex vivo expansion can result in T cell exhaustion, which reduces the effector function. There is also a batch-to-batch variation during the manufacturing process, making it difficult to standardize and validate the cell products. In addition, the process is labor-intensive and costly. Generation of universal off-the-shelf CAR T cells, which can be broadly given to any patient, prepared in advance and ready to use, would be ideal and more cost-effective. Human induced pluripotent stem cells (iPSCs) provide a renewable source of cells that can be genetically engineered and differentiated into immune cells with enhanced anti-tumor cytotoxicity. This review describes basic knowledge of T cell biology, applications in ACT, the use of iPSCs as a new source of T cells and current differentiation strategies used to generate T cells as well as recent advances in genome engineering to produce next-generation off-the-shelf T cells with improved effector functions. We also discuss challenges in the field and future perspectives toward the final universal off-the-shelf immunotherapeutic products

CRISPR-Cas9-mediated disruption of B2M and CIITA genes eliminates HLA class I and II expression in human induced pluripotent stem cells (MUSIi001-A-2)

Cell-based therapy offers great promise for treating degenerative diseases. Although autologous cell-based therapy is ideal, it may be impractical due to the high manufacturing cost and long production time. Allogeneic cell-based therapy offers a more cost-effective alternative; however, the risk of graft rejection is a major concern. Here, we generated HLA class-I and -II null iPSC line by knocking out CIITA gene in the B2M-knockout MUSIi001-A-1 cell line using CRISPR/Cas9 system. The MUSIi001-A-2 line provides a valuable model for studying immunological responses against allogeneic T cells and serves as a prototype for developing specific cell types for future cell-based therapy

Establishment of a human iPSC line (MUSIi007-A) from peripheral blood of normal individual using Sendai viral vectors

Human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMNCs) isolated from a 26-year-old healthy subject to use as a control group for the iPSC line carrying compound heterozygote for mutation in KLF1 gene. The cells were reprogrammed using integration-free method, Sendai viral (SeV) vectors containing KOS, hc-MYC and hKLF4. The established iPSC line (MUSIi007-A) exhibited a normal karyotype, expressed pluripotent markers and displayed in vitro and in vivo differentiation potential into cells of three embryonic germ layers.Resource tableUnlabelled TableUnique stem cell line identifierMUSIi007-AAlternative name(s) of stem cell lineSeV-iPSCInstitutionSiriraj Center of Excellence for Stem Cell Research,Faculty of Medicine Siriraj Hospital, Mahidol UniversityContact information of distributorPonthip Potirat, [email protected] Issaragrisil, [email protected] of cell lineiPSCsOriginHumanAdditional origin infoAge: 26-year-oldSex: MaleEthnicity: ThaiCell sourcePeripheral blood mononuclear cellsClonalityClonalMethod of reprogrammingIntegration-free Sendai viral vectorsGenetic modificationN/AType of modificationN/AAssociated diseaseN/AGene/locusN/AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateDecember 2014Cell line repository/bankN/AEthical approvalSiriraj Institutional Review Board (SiRB), no. Si248/2011, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand

Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication

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

Generation of two induced pluripotent stem cell lines (MUSIi011-A and MUSIi011-B) from peripheral blood T lymphocytes of a healthy individual

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

An integration-free iPSC line (MUSIi008-A) derived from a patient with severe hemolytic anemia carrying compound heterozygote mutations in KLF1 gene for disease modeling

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

Establishment of an integration-free induced pluripotent stem cell line (MUSIi005-A) from exfoliated renal epithelial cells

Human induced pluripotent stem cells (iPSCs) were generated from exfoliated renal epithelial cells isolated from a urine sample of a 31-year-old healthy woman. Epithelial cells were characterized for the expression of E-cadherin and reprogrammed using non-integrating Sendai viral vectors. The urine-derived iPSC line (designated as MUSIi005-A) was karyotypically normal, expressed pluripotent markers, differentiated into cells of three embryonic germ layers, and showed no viral and transgene expressions at passage 29. Our protocol offers a non-invasive and efficient approach for iPSC generation from patients with genetic or acquired disorders