Table 1. Methods for stem cell reprogramming. Current approaches to reprogramming somatic cells into induced pluripotent stem (iPS) cells fall into four major categories, each using a different set of vectors and/or molecules to deliver reprogramming factors into the cells. Sources: Anokye-Danso, F. et al. Cell Stem Cell 8, 376-388 (2011). Ho, R. et al. J. Cell Phys. 226, 868-878 (2011). Nishimura, K. et al. J. Biol. Chem. 286, 4760-4771 (2011). Stadtfeld, M. & Hochedlinger, K. Genes Dev. 24, 2239-2263 (2010). BioCentury Archives

Delivery/
Expression system

Description

Pros

Cons

Category: Integrating/nonexcisable

Retrovirus

Somatic cells are transduced with retrovirus encoding genes for reprogramming factors

Average efficiency

Transgenes for reprogramming factors are silenced after reprogramming into iPS cell state

Genomic integration

Transgene silencing may be incomplete and interfere with subsequent differentiation steps

Lentivirus

Somatic cells are transduced with lentivirus encoding genes for reprogramming factors

Average efficiency

Transgenes are silenced after reprogramming

Genomic integration

Transgene silencing is less efficient than retrovirus

Inducible lentivirus

Somatic cells are transduced with lentivirus encoding inducible genes for reprogramming factors

Transgene expression in infected cells is induced with inert drugs (for example, doxycycline)

Average efficiency

Controlled transgene expression

Genomic integration

Secondary inducible lentiviral system

Primary populations of iPS cells are generated with an inducible lentivirus system

Primary iPS cells are then differentiated into somatic cells that still carry the inducible transgenes

Inert drug is then used to induce transgene expression in differentiated somatic cells to generate secondary iPS cells

Average to very high efficiency depending on cell type

Controlled transgene expression

No direct delivery of virus to secondary iPS cell population

Capable of reprogramming cells that are difficult to transduce

Genomic integration

Requires additional steps to differentiate and screen cells

Category: Integrating/excisable

Lentivirus with floxed transgenes

Somatic cells are transduced with a lentivirus encoding excisable genes for reprogramming factors

Virus-transduced transgenes are excised from the cell genome with a Cre recombinase when they are no longer needed

Average efficiency

Transgenes removed

Requires additional steps to screen and analyze cells

Short sequences from vector still remain in genome

Transposon

Transposon (for example, piggyBac) encoding genes for reprogramming factors are introduced into somatic cell genome using transposase

Transposase also used to excise transposon from cell genome when transgenes are no longer needed

Average efficiency

All vector sequences removed

Requires additional steps to screen and analyze cells

Category: Nonintegrating/DNA based

Adenovirus

Somatic cells are transduced with an adenovirus encoding genes for reprogramming factors

No genomic integration under normal circumstances

Low efficiency

Potential for vector DNA to integrate with host cell genome is low but still exists

Requires additional steps to screen and analyze cells for possible vector integration

Plasmid

Somatic cells are transfected with a plasmid encoding genes for reprogramming factors

Episome

Somatic cells are transfected with episomes encoding genes for reprogramming factors

Minicircle

Somatic cells are transfected with a minicircle encoding genes for reprogramming factors

Category: Nonintegrating/DNA free

Protein

Reprogramming factors delivered directly into somatic cells

Delivered as purified recombinant proteins or as whole-cell extracts from embryonic stem cells (ESCs), genetically engineered human cells or bacteria

No genomic integration

Low efficiency

Need for steady supply of reprogramming factors can become expensive

Sendai virus

Somatic cells are transduced with Sendai virus encoding genes for reprogramming factors

Vector is RNA and thus will not integrate into host cell genome

Vector replicates in host cell cytoplasm

High efficiency

No genomic integration

Reprogramming factors produced in high quantities

Reprogrammed cells need to be continuously passaged to remove virus-encoded transgenes

Modified mRNA

Somatic cells are transfected with modified mRNAs encoding reprogramming factors

High efficiency

No genomic integration

Requires multiple rounds of transfection

MicroRNAA

Somatic cells are transfected with miRNAs

Reprograms somatic cells without exogenous, transcription factor-based reprogramming agents

May have high efficiency

May be able to reprogram somatic cells that are refractory to reprogramming with standard reprogramming factors

Method still needs to be replicated and validated with an existing nonintegrating method for miRNA delivery

Mechanism of reprogramming still needs to be defined

AStudy used integrating lentivirus vector to deliver miRNAs, but nonintegrating vectors for delivering miRNA into cells already exist and have been used to increase iPS cell reprogramming efficiency with transcription factors.