Induced Pluripotent Stem Cells (iPSCs)

Initially described in the pioneering work of Yamanaka and colleagues, the ability to "reprogram" differentiated somatic cells into a pluripotent embryonic stem cell-like state by retroviral mediated expression of four specific transcription factors has revolutionized our ability to develop new models to study human disease and represents a significant step towards patient-specific cell replacement therapies.

What can I do with iPSCs?

In addition to solving ethical concerns related to the use of blastocyst-derived embryonic stem cells, the use of iPSCs for the generation of therapeutic cells for cell replacement therapy may avoid the requirement for post-transplant immune suppression because iPSCs can be generated directly from the transplant recipient and will therefore be genetically identical to the patient. Additionally, because it is possible to reprogram somatic cells derived from diseased individuals iPSC technology provides an important new platform for the development of new models of human disease. Thus, upon appropriate differentiation these cells can then be used to study normal and pathologic human tissue development in vitro, enabling new insights into disease pathology as well as a platform for the development of novel therapeutic agents and patient-specific cell replacement therapies.

iPSC technologies used in the PSCF

  • We have successfully generated iPSCs from numerous human cell types derived from both patients and healthy individuals. Cell types successfully reprogrammed include: fibroblasts, peripheral blood mononuclear cells, keratinocytes, nasal epithelial cells, urine-derived epithelial cells and CD34+ bone marrow and cord blood cells.
  • The PSCF evaluates novel reprogramming methods as they become available to ensure we are using the most efficient and cost-effective methods.
  • Currently, the PSCF uses an excisable, polycistronic lentiviral vector to generate high quality iPSCs from peripheral blood mononuclear cells with high efficiency.
  • We also use a non-integrating, episomal plasmid-based method to generate high quality, integration-free iPSCs from fibroblasts.
  • Using these methods, we have successfully generated high-quality iPSCs from fibroblasts and peripheral blood mononuclear cells from hundreds of individuals.

iPSC services available in the PSCF

The PSCF is committed to facilitating access to this exciting technology for Cincinnati Children's Hospital Medical Center/UC researchers.
We provide the following iPSC services:

  • iPSC generation on a fee-for-service basis
  • iPSC characterization and quality control assays
  • Training in iPSC generation (Cincinnati Children's/UC investigators only)

Click here for current pricing information.

Download typical iPSC generation flow chart with time line here.

Characterization / quality control of iPSCs

Characterizing iPSC lines using a standard series of quality control metrics is critical before investing resources in project-specific iPSC analyses. The PSCF has implemented all the standard assays used for basic iPSC quality assessment.

Morphology

Typical hESC-like morphology of an iPSC line derived from human fibroblasts using non-integrating episomal plasmids. These cells have been cultured in feeder-free culture conditions for 15 passages. Note the identical colony and cellular morphology compared to H1 hESCs cultured under these conditions.

Pluripotency Marker Expression
iPSC lines derived from fibroblasts or cord blood using 4 separate retroviruses or a polycistronic lentivirus respectively were fixed with 4% paraformaldehyde and the expression of pluripotency markers Tra-1-60 and Dnmt3b were assessed by immunofluorescence analysis. Note identical staining patterns compared to H1 hESCs.

In-Vitro Embryoid Body and In-Vivo Teratoma Differentiation

In-vivo differentiation of PBMC-iPSCs in teratomas. iPSCs derived from fibroblasts using episomal plasmids were injected subcutaneously into immunodeficient (NOD/Shi-scid/IL-2Rγnull) mice. After ~10 weeks, teratomas were excised and histology was analyzed by hematoxylin/eosin staining. Differentiation of iPSCs into tissues derived from each embryonic germ layer was confirmed in H&E stained slides by the presence of glandular epithelia (endoderm), neural rosettes (ectoderm), and cartilage (mesoderm).

Karyotype Analysis
G-banded karyotype analysis of PBMC-iPSCs demonstrating normal karyotype.

Confirmation of Genetic Identity
We recommend that STR analysis is used to confirm the identity of iPSCs following generation (e.g. by comparison to donor cells) and during routine culture.

Directed Differentiation 
We have successfully performed directed differentiation of iPSC lines into numerous cell types including intestine, neural progenitors and cardiomyocytes. Please see our Differentiation page for details.

Genomic iPSC Characterization
Additional iPSC characterization metrics focusing on genomic analyses (e.g. RNA-seq, micro-RNA, methylation analysis, DNA fingerprinting, copy number variation) are available through the Cincinnati Children's Hospital Medical Center Genetic Variation and Gene Discovery Core Facility.

iPSC line banking
iPSC lines generated by the PSCF (for Cincinnati Children's/UC investigators only) can be stored securely in liquid nitrogen in the Cincinnati BioBank Core.