A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease-Specific Induced Pluripotent Stem Cells.

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, ß-globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation.

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation.

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.

Generation of human induced pluripotent stem cells from peripheral blood using the STEMCCA lentiviral vector.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)(1-4). Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies(5). We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors(6). These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs(7-9). In one study, a Tet inducible version of the STEMCCA vector was employed(9), which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies. Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described(10,11) in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.