ABSTRACT
A patient with ß(E)/ß(0) -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, ß-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSC-derived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the ß-hemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with ß-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration.
Subject(s)
Globins/genetics , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Lentivirus/metabolism , Transduction, Genetic , beta-Thalassemia/pathology , Adult , Animals , Cell Differentiation/drug effects , Erythroid Cells/cytology , Erythroid Cells/drug effects , Erythroid Cells/metabolism , Gene Expression Regulation/drug effects , Genetic Vectors/metabolism , Globins/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Mice , Mutagens/toxicity , Regeneration/drug effects , Virus Integration/drug effectsABSTRACT
Understanding the steps and cues that allow hematopoietic cells to be generated during development holds great clinical as well as biological interest. Analysis of these events in mice has provided many important insights into the processes involved, but features that might be unique to humans remain challenging to elucidate because they cannot be studied directly in vivo. Human embryonic stem or induced pluripotent stem cells offer attractive in vitro alternatives to analyze the process. Here we review recent efforts to develop defined and quantitative systems to address outstanding developmental questions against a background of what we know about the development of hematopoietic cells in the fetus and derived from mouse embryonic stem cells.
Subject(s)
Hematopoiesis , Hematopoietic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation , Embryonic Stem Cells/cytology , Humans , Mesoderm/physiology , Models, BiologicalABSTRACT
Realizing the potential that human embryonic stem cells (hESCs) hold, both for the advancement of biomedical science and the development of new treatments for many human disorders, will be greatly facilitated by the introduction of standardized methods for assessing and altering the biological properties of these cells. The 7-day in vitro alkaline phosphatase colony-forming cell (AP(+)-CFC) assay currently offers the most sensitive and specific method to quantify the frequency of undifferentiated cells present in a culture. In this regard, it is superior to any phenotypic assessment protocol. The AP(+)-CFC assay, thus, provides a valuable tool for monitoring the quality of hESC cultures, and also for evaluating quantitative changes in pluripotent cell numbers following manipulations that may affect the self-renewal and differentiation properties of the treated cells. Two other methods routinely used to evaluate hESC pluripotency involve either culturing the cells under conditions that promote the formation of nonadherent differentiating cell aggregates (termed embryoid bodies), or transplanting the cells into immunodeficient mice to obtain teratomas containing differentiated cells representative of endoderm, mesoderm, and ectoderm lineages.
Subject(s)
Colony-Forming Units Assay/methods , Embryonic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Alkaline Phosphatase/metabolism , Animals , Cell Differentiation/drug effects , Cell Line , Collagen/pharmacology , Drug Combinations , Embryoid Bodies/cytology , Embryoid Bodies/drug effects , Embryoid Bodies/metabolism , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/enzymology , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Injections, Subcutaneous , Laminin/pharmacology , Mice , Mice, SCID , Neoplasm Transplantation , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/enzymology , Proteoglycans/pharmacology , Teratoma/pathology , Tumor Stem Cell AssayABSTRACT
OBJECTIVE: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells, although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells, a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives, including hematopoietic cells, from hESCs and hiPSCs. MATERIALS AND METHODS: Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays, flow cytometry, quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor gamma-chain-null mice for almost 1 year. RESULTS: Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages, and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor gamma-chain-null mice. CONCLUSIONS: MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Lineage , Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Animals , Carcinoma, Hepatocellular/pathology , Cell Differentiation , Coculture Techniques , Culture Media, Conditioned/pharmacology , Humans , Mice , Paracrine CommunicationABSTRACT
Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of >or=30 AP(+) cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable, and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.
Subject(s)
Alkaline Phosphatase/metabolism , Cell Differentiation , Colony-Forming Units Assay , Embryonic Stem Cells/cytology , Embryonic Stem Cells/enzymology , Animals , Antigens, Tumor-Associated, Carbohydrate/metabolism , Cell Count , Cell Line , Cell Proliferation , Clone Cells , Glycosphingolipids/metabolism , Humans , Mice , Phenotype , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/enzymology , Sensitivity and Specificity , Stage-Specific Embryonic AntigensABSTRACT
Chromatin remodeling complexes play critical roles in development. Here we describe a transcription factor, CECR2, which is involved in neurulation and chromatin remodeling. CECR2 shows complex alternative splicing, but all variants contain DDT and bromodomain motifs. A mutant mouse line was generated from an embryonic stem cell line containing a genetrap within Cecr2. Reporter gene expression demonstrated Cecr2 expression to be predominantly neural in the embryo. Mice homozygous for the Cecr2 genetrap-induced mutation show a high penetrance of the neural tube defect exencephaly, the human equivalent of anencephaly, in a strain-dependent fashion. Biochemical isolation of CECR2 revealed the presence of this protein as a component of a novel heterodimeric complex termed CECR2-containing remodeling factor (CERF). CERF comprises CECR2 and the ATP-dependent chromatin remodeler SNF2L, a mammalian ISWI ortholog expressed predominantly in the central nervous system. CERF is capable of remodeling chromatin in vitro and displays an ATP hydrolyzing activity that is stimulated by nucleosomes. Together, these data identify a novel chromatin remodeling complex with a critical role in neurulation.
Subject(s)
Central Nervous System/embryology , Chromatin Assembly and Disassembly , Chromatin , DNA-Binding Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Alternative Splicing , Animals , Cells, Cultured , Central Nervous System/metabolism , DNA-Binding Proteins/genetics , Female , Intercellular Signaling Peptides and Proteins/genetics , Male , Mice , Mice, Inbred BALB C , Mutation , Neural Tube Defects , Nucleosomes/metabolism , Transcription Factors/geneticsABSTRACT
Pericentromeric regions of human chromosomes are preferential sites for the integration of duplicated DNA, or "duplicons", which often contain gene fragments. Although pericentromeric regions appear to be genomic junkyards, they could also be the birthplace of new genes with novel functions. We have characterized a chimeric transcription unit (cat eye syndrome critical region gene 7, CECR7) formed from three duplicons in the pericentromeric region of chromosome 22q. CECR7 exons show similarity to sequences on chromosomes 2, 5, 7, 10, 11, 12, 13, 14, 15, 16, 18, 19, 21, and elsewhere on 22. Based on polymerase chain reaction (PCR) analysis of CECR7 duplicon boundaries in various primate species, and the sequence divergence between the human duplicons and their putative ancestral human loci, CECR7 was probably formed before the separation of macaque and is therefore older than most previously reported pericentromeric duplicons. Expression of CECR7 was detected by RT-PCR in humans and gorilla fibroblasts, but not orangutan, suggesting that expression did not result immediately from the formation of this novel transcription unit, or that expression was silenced in orangutan following its formation.