Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases
Abstract
:1. Introduction
1.1. Stem Cells: Features
1.2. Stem Cells: Source for Potential Therapy
2. Induced Pluripotent Stem Cells: A Novel Solution to an Old Problem
3. Generation of iPSCs
3.1. Moloney-Based Retrovirus
3.2. HIV-Based Lentivirus
3.3. Transient Transfection and Adenovirus
3.4. Small Molecules
3.5. Protein Transduction
3.6. Genome Editing
4. iPSCs and Hematopoiesis: Are We There Yet?
4.1. Red Blood Cells/Platelets
4.2. Neutrophils and Monocytes/Macrophages
4.3. Dendritic cells and Natural Killer (NK) Cells
4.4. T and B Lymphocytes
4.5. Hematopoietic Stem Cells
5. iPSCs Hematological Disorders
5.1. iPSCs in Congenital Hematopoietic Diseases
5.1.1. Fanconia Anemia
5.1.2. β-Thalassemia
5.2. Generation of iPSCs from Hematologic Malignancy
5.2.1. Myeloproliferative Neoplasms
5.2.2. Myelodysplastic Syndrome
6. Conclusions
Acknowledgments
Conflicts of Interest
References
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Embryonic Stem Cells (ESCs) | Hematopoietic Stem Cells (HSCs) | Induced Pluripotent Stem Cells (iPSCs) | |
---|---|---|---|
Source | Inner mass cells of blastocyst | Bone marrow donations; umbilical cord blood | Any somatic cell |
Application | Basic science research; limited clinical application currently | Hematopoietic stem cell transplantation | Basic science research |
Markers | SOX2, NANOG, Oct-4, SSEA-1, SSEA-3, SSEA-4 TRA-1-60, TRA-1-81 Frizzled5 | CD34+, c-Kit−/low, Lin-, CD38-,Flt-3/Flk-2 | Reactivation of embryonic stem cell markers, e.g., SOX2, NANOG, OCT-4, KLF4, SSEA-4, TRA-1-60 |
Derivation | Isolation from in vitro fertilized embryos | Purification fromdonations | Ectopic expression of ESC transcription factors: OCT3/4, SOX2, KLF4, c-MYC |
Pros | Able to generate all three germ layers; Amenable to cell culture expansion while maintaining pluripotency | Not controversial; Can be harvested from bone marrow blood, can be mobilized to peripheral blood upon granulocyte-colony stimulating factor (G-CSF) induction, or obtained from umbilical cord blood donations; Rise in biobanking of umbilical cord blood increases amount of source material | Non-invasive isolation; Avoids Human Leukocyte Antigen Loci (HLA)-compatibility issues Can be genetically altered before transfusion; Expands available research areas; Recapitulates patient genome; Theoretically unlimited source material |
Cons | Ethical concerns of using embryonic-derived cells; Limited source material | Restricted lineage differentiation; Dependent on HLA compatibility; Transplantation-associated risks: immune suppression, graft rejection, graft vs. host disease | Low efficiency of reprogramming;Incomplete programming, or “epigenetic memory”; No standardized protocol for production; Genetically unstable; Safety concerns; Maintenance of germline mutations; Insertional mutagenesis for integrating vectors |
Induced Pluripotent Stem Cells (iPSCs) Protocol | Consideration | |
---|---|---|
1. Choice of Cell Type | Adult mouse and human fibroblasts were used in first iPSCs experiments. | While iPSCs can in principle be generated from any somatic cell, in practice, there seems to be an inverse relationship between degree of differentiation and ease of reprogramming. Additionally, there is expanding concern for “memory” of the original cell, hindering the re-differentiation process downstream. |
2. Dedifferentiation | Retroviral- or lentiviral-mediated expression of four pluripotent-specific genes: OCT3/4, SOX2, KLF4, and c-MYC (OSKM). | Concern for the transforming potential of c-MYC led to the identification of other factor substitutes. c-MYC was later deemed dispensible, and other factor combinations (Nanog, Lin28) have successfully generated iPSCs.Methods of delivery must also consider the effects of insertional mutagenesis when using integrating vectors. Non-integrating viruses, small-molecules, RNA- and transposon-based technologies are also currently being explored. |
3. Selection | Transduced cells are cultured in embryonic stem cell (ESC) medium + antibiotics for 2–4 weeks with an ESC-specific marker, Fbx15, driving antibiotic resistance. Only reprogrammed cells can survive the selection process. | Although Fbx15 is expressed only in ESCs, it is not essential to ESC development and explains the partial reprogramming observed initially. Currently, Nanog-driven selection is favored instead. |
4. Differentiation | Cultured with feeder cells and cytokines directing lineage-specific differentiation. | iPSCs can differentiate through (direct) addition of lineage-specific transcription factors or (indirect) culture in lineage-specific cytokines and growth factors. Protocols vary among laboratories. |
5. Functional Testing | Expression of lineage-specific markers measured through PCR or immunofluorescence. | Functional tests are not standardized. Definition of lineage-specific characteristics vary among laboratories. |
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Li, M.; Cascino, P.; Ummarino, S.; Di Ruscio, A. Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases. Cells 2017, 6, 7. https://doi.org/10.3390/cells6010007
Li M, Cascino P, Ummarino S, Di Ruscio A. Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases. Cells. 2017; 6(1):7. https://doi.org/10.3390/cells6010007
Chicago/Turabian StyleLi, Mailin, Pasquale Cascino, Simone Ummarino, and Annalisa Di Ruscio. 2017. "Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases" Cells 6, no. 1: 7. https://doi.org/10.3390/cells6010007
APA StyleLi, M., Cascino, P., Ummarino, S., & Di Ruscio, A. (2017). Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases. Cells, 6(1), 7. https://doi.org/10.3390/cells6010007