Tools: Finding a home for nuclear transfer 

 

Table 1. Stem cell types and methods for stem cell generation. There are three major classes of stem cells: embryonic stem cells (ESCs), induced pluripotent stem (iPS) cells and tissue-specific stem cells. Each class has distinct advantages and drawbacks, including how the cells are generated or extracted.

Source: Refs. 1, 8-12; BCIQ: BioCentury Online Intelligence; California Institute of Regenerative Medicine; EuroStemCell

 

Advantages

Drawbacks

ESCs

- Can differentiate into any cell type
- Can self-renew indefinitely

- Number of lines available is limited
- Derivation requires the use of donor oocytes
-
Many older lines are unsuitable for therapeutic use owing to contamination

- Use carries risk of teratoma
- Cells derived from ESCs may not recapitulate adult cell phenotype
- Use faces major ethical, regulatory and funding obstacles

Methods for generating ESCs

Derivation from in vitro fertilized embryos

- Easier to apply than nuclear transfer
- Protocols are well established
- Therapeutic candidates have entered clinical trials

- Donor and recipient cells are genetically distinct

Somatic cell nuclear transfer

-
Genetically matched to somatic cell donor, except for mitochondrial DNA

- Uses unfertilized oocytes

- Methods are technically cumbersome
- Protocols need further optimization
- Scalability and efficiency of approach still need to be determined

iPS cells

- Can differentiate into any cell type
- Can self-renew indefinitely
- Donor and recipient cells can be genetically matched
- Source cells are plentiful and easy to obtain
- Reprogramming protocols are highly scalable
- Use faces fewer ethical and funding obstacles than with ESCs

- Use carries risk of teratoma
-
Cells derived from iPS cells may not recapitulate adult cell phenotype

- Immunogenicity is possible even if cells are genetically matched
- iPS cell-derived cell therapies have not yet entered clinical trials
- Therapeutic development assumed to carry higher risk than ESCs

Methods for generating iPS cells 

Generated with integrating, nonexcisable DNA-based vectors

-
Reprogramming efficiency is average to high, depending on vector

-
Reprogramming factor transgenes are silenced after reprogramming step

-
Some vectors (such as inducible lentivirus) use inducible transgene expression systems to provide fine control of reprogramming factor expression

- Genomic integration raises additional safety concerns
- Transgene silencing may be incomplete

Generated with integrating, excisable DNA-based vectors

- Reprogramming efficiency is average
-
Transgenes are removed from host cell genome after reprogramming step

- Additional steps are needed to confirm transgene removal in cells
-
Some vectors (such as lentivirus with floxed transgenes) still leave sequences in host cell genome

Generated with nonintegrating DNA-based vectors

-
Genomic integration does not occur under normal circumstances

- Reprogramming efficiency is low
-
Vector DNA still has a low potential to integrate with host cell genome

-
Additional steps are needed to check for possible genomic integration

Generated with nonintegrating RNA-based vectors

- Reprogramming efficiency is high
- Genomic integration does not occur
-
Some vectors (such as Sendai virus) can stimulate very high levels of reprogramming factor production

-
Some vectors (such as microRNAs) might be able to reprogram somatic cells refractory to other reprogramming approaches

-
Replicating viral vector must be removed after reprogramming step

-
Reprogramming with nonviral vectors may require multiple rounds of transfection

Generated with proteins and/or small molecule cocktails

- Genomic integration does not occur

- Reprogramming efficiency is low
-
Need for constant supply of reprogramming factors can be expensive

Tissue-specific stem cells

- Marketed therapies have been shown not to cause tumors
-
Approved therapies that contain tissue-specific stem cells already exist

- Cells usually recapitulate adult cell phenotype
- Certain types (such as umbilical) can be frozen and stored
-
Use faces few ethical and funding obstacles compared with ESCs

- Cells can differentiate into a limited number of cell types
- Capacity for self-renewal is limited
- Cells are present in small quantities in source tissues
- Cells are less scalable than iPS cells

Methods for generating tissue-specific stem cells

Extraction from autologous tissues

- Cells are genetically matched to the patient
- Protocols are well established

- Autologous source tissues are limited in supply

Extraction from allogeneic tissues

-
Allogeneic source tissues may be more plentiful than autologous tissues

-
Method could enable the development of off-the-shelf therapies

- Protocols are well established

- Donor and recipient cells are genetically distinct