Induced pluripotent stem cell

Induced pluripotent stem cells^[1], commonly abbreviated as iPS cells or iPSCs, are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inserting certain genes.

Induced Pluripotent Stem Cells are believed to be identical to natural pluripotent stem cells, such as embryonic stem cells in many respects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability, but the full extent of their relation to natural pluripotent stem cells is still being assessed.

IPSCs were first produced in 2006 from mouse cells and in 2007 from human cells. This has been cited as an important advancement in stem cell research, as it may allow to obtain pluripotent stem cells, which are important in research and potentially have therapeutic uses, without the controversial use of embryos.

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Production of iPSCs

  iPS cells are typically derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved through viral vectors, such as retroviruses. Transfected genes include the master transcriptional regulators Oct-3/4 (Pouf51) and Sox2, although it is suggested that other genes enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic infection.

First generation

Induced pluripotent stem cells were first generated by Shinya Yamanaka's team at Kyoto University, Japan in 2006. Yamanaka had identified genes that are particularly active in embryonic stem cells, and used retroviruses to transfect mouse fibroblasts with a selection of those genes. Eventually, four key pluripotency genes essential for the production of pluripotent stem cells were isolated; Oct-3/4, Sox2, c-Myc, and Klf4. Cells were isolated by antibiotic selection for Fbx15⁺ cells. However, this iPS line showed DNA methylation errors compare to original pattern in ESC line and failed to produce viable chimeras if injected into developing embryos.

Second generation in mice

In June 2007, the same group published a breakthrough study along with two other independent research groups from Harvard, MIT, and the University of California, Los Angeles, showing successful reprogramming of mouse fibroblasts into iPS and even producing viable chimera. These cell lines were also derived from mouse fibroblast by retroviral mediated reactivation of the same four endogenous pluripotent factors, but the researchers now selected a different marker for detection. Instead of Fbx15, they used Nanog which is an important gene in ESCs. DNA methylation patterns and producing viable chimeras (and thereby contributing to subsequent germ-line production) indicated that Nanog is a major determinant of cellular pluripotency.^{[2] [3] [4] [5] [6]}

Unfortunately, one of the four genes used (namely, c-Myc) is oncogenic, and 20% of the chimeric mice developed cancer. In a later study, Yamanaka reported that one can create iPSCs even without c-Myc. The process takes longer and is not as efficient, but the resulting chimeras didn't develop cancer.^[7]

Human induced pluripotent stem cells

In November 2007, a milestone was achieved^[8][1] by creating iPS from the adult human cells by two independent research teams' studies which were released - one in Science by James Thomson and colleagues at University of Wisconsin-Madison^[9] and second in Cell by Shinya Yamanaka and colleagues at Kyoto University, Japan^[10]. With the same principle used earlier in mouse models, Yamanaka had successfully transformed human fibroblasts into pluripotent stem cells using the same four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc with a retroviral system. Thomson and colleagues used OCT4, SOX2, NANOG, and a different gene LIN28 using a lentiviral system.

The viral transfection systems used insert the genes at random locations in the host's genome; this is a concern for potential therapeutic applications of these iPSCs, because the created cells might be susceptible to cancer. Members of both teams consider it therefore necessary to develop new delivery methods.^[7]

Identity

The generated iPSCs were remarkably similar to naturally-isolated pluripotent stem cells (such as mouse and human embryonic stem cells, mESCs and hESCs, respectively) in the following respects, thus confirming the identity, authenticity, and pluripotency of iPSCs to naturally-isolated pluripotent stem cells:

• Cellular biological properties
  • Morphology: iPSCs were morphologically similar to ESCs. Each cell had round shape, large nucleolus and scant cytoplasm. Colonies of iPSCs were also similar to that of ESCs. Human iPSCs formed sharp-edged, flat, tightly-packed colonies similar to hESCs and mouse iPSCs formed the colonies similar to mESCs, less flatter and more aggregated colonies than that of hESCs.
  • Growth propeties: Doubling time and mitotic activity are cornerstones of ESCs, as stem cells must self-renew as part of their definition. iPSCs were mitotically active, actively self-renewing, proliferating, and dividing at a rate equal to ESCs.
  • Stem Cell Markers: iPSCs expressed cell surface antigenic markers expressed on ESCs. Human iPSCs expressed the markers specific to hESC, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Mouse iPSCs expressed SSEA-1 but not SSEA-3 nor SSEA-4, similarly to mESCs.
  • Stem Cell Genes: iPSCs expressed genes expressed in undifferentiated ESCs, including Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.
  • Telomerase Activity: Telomerases are necessary to sustain cell division unrestricted by the Hayflick limit of ~50 cell divisions. hESCs express high telomerase activity to sustain self-renewal and proliferation, and iPSCs also demonstrate high telomerase activity and express hTERT (human telomerase reverse transcriptase), a necessary component in the telomerase protein complex.
• Pluripotency: iPSCs were capable of differentiation in a fashion similar to ESCs into fully differentiated tissues.
  • Neural Differentiation: iPSCs were differentiated into neurons, expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B, and MAP2. The presence of catecholamine-associated enzymes may indicate that iPSCs, like hESCs, may be differentiable into dopaminergic neurons. Stem cell-associated genes were downregulated after differentiation.
  • Cardiac Differentiation: iPSCs were differentiated into cardiomyocytes that spontaneously began beating. Cardiomyocytes expressed TnTc, MEF2C, MYL2A, MYHCβ, and NKX2.5. Stem cell-associated genes were downregulated after differentiation.
  • Teratoma Formation: iPSCs injected into immunodeficient mice spontaneously formed teratomas after nine weeks. Teratomas are tumors of multiple lineages containing tissue derived from the three germ layers endoderm, mesoderm and ectoderm; this is unlike other tumors, which typically are of only one cell type. Teratoma formation is a landmark test for pluripotency.
  • Embryoid Body: hESCs in culture spontaneously form ball-like embryo-like structures termed “embryoid bodies”, which consist of a core of mitotically active and differentiating hESCs and a periphery of fully differentiated cells from all three germ layers. iPSCs also form embryoid bodies and have peripheral differentiated cells.
  • Blastocyst Injection: hESCs naturally reside within the inner cell mass (embryoblast) of blastocysts, and in the embryoblast, differentiate into the embryo while the blastocyst’s shell (trophoblast) differentiates into extraembryonic tissues. The hollow trophoblast is unable to form a living embryo, and thus it is necessary for the embryonic stem cells within the embryoblast to differentiate and form the embryo. iPSCs were injected by micropipette into a trophoblast, and the blastocyst was transferred to recipient females. Chimeric living mouse pups were created: mice with iPSC derivatives incorporated all across their bodies with 10%-90& chimerism.
• Epigenetic reprogramming
  • Promoter Demethylation: Methylation is the transfer of a methyl group to a DNA base, typically the transfer of a methyl group to a cytosine molecule in a CpG site (adjacent cytosine/guanine sequence). Widespread methylation of a gene interferes with expression by preventing the activity of expression proteins or recruiting enzymes that interfere with expression. Thus, methylation of a gene effectively silences it by preventing transcription. Promoters of pluripotency-associated genes, including Oct-3/4, Rex1, and Nanog, were demethylated in iPSCs, demonstrating their promoter activity and the active promotion and expression of pluripotency-associated genes in iPSCs.
  • Histone Demethylation: Histones are compacting proteins that are structurally localized to DNA sequences that can effect their activity through various chromatin-related modifications. H3 histones associated with Oct-3/4, Sox2, and Nanog were demethylated, indicating the expression of Oct-3/4, Sox2, and Nanog.

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