Biomedical Engineering Reference
In-Depth Information
using hESCs, however, as embryos are destroyed in order to obtain these cells. Therefore, use
of hESCs is currently banned in many countries.
Stem Cells from Somatic Cell Nuclear Transfer
Cloning procedures provide another source of stem cells for tissue engineering; moreover,
tremendous interest has been generated in the field of nuclear cloning since the 1997 birth of
a cloned sheep named Dolly, which was the first mammal to be derived from an adult somatic
cell by use of nuclear transfer [47]. Since then, animals from several species have been grown
using nuclear transfer technology, including cattle [48], goats [49], mice [50], and pigs [51, 52].
Reproductive cloning and therapeutic cloning are two types of nuclear cloning [53, 54]. In
reproductive cloning, an embryo is generated with the identical genetic material as its cell
source. The embryo can then be implanted into the uterus of a donor to give rise to an off-
spring that is a clone of the cell donor; however, this type of cloning is banned in most coun-
tries for human applications. For therapeutic cloning, early-stage embryos are grown in
culture to produce embryonic stem-cell lines that contain identical genetic material to that of
their source. These autologous stem cells would be beneficial in tissue and organ replacement
applications because they are capable of differentiating to a variety of cell types in the adult
body [55] and may provide an alternate source of allogeneic transplantation, which would
eliminate rejection due to immunologic incompatibility, and use of immunosuppressive
drugs [55]. Somatic cell nuclear transfer technology has certain limitations that require
further study before this technique can be applied clinically. Cloning efficiency is very low, as
most embryos derived from the cloning process do not survive [56-58]. Refinement of the
complex steps of nuclear transfer, as well as cell cycle synchronization between donor cells
and recipient oocytes, must be accomplished to improve cloning efficiency [59].
Reprogramming and Generation of Induced Pluripotent State Cells
Reports of the successful transformation of adult somatic cells into pluripotent stem cells
(PSCs) through genetic “reprogramming” have been published in recent years. Reprogramming
involves de-differentiation of adult somatic cells (such as fibroblasts) to produce patient-
specific pluripotent stem cells that are genetically identical to the donor somatic cells, which
eliminates rejection. Yamanaka's group was the first to discover that mouse embryonic fibro-
blasts and adult mouse fibroblasts could be reprogrammed into an “induced pluripotent
state” [60]. Of the 24 genes they examined that were thought to be important for embryonic
stem cells, they identified four key genes: Oct3/4, Sox2, c-Myc, and Klf4. The induced plurip-
otent state (iPS) cells generated by this reprogramming technique possessed the immortal
growth characteristics of self-renewing ESCs, expressed genes specific for ESCs, and gener-
ated embryoid bodies
in vitro
and teratomas
in vivo
[60]. When iPS cells were injected into
mouse blastocysts, they contributed to a variety of cell types; however, iPS cells selected
using this method were pluripotent, they were not identical to ESCs [60]. Chimeras made
from iPS cells did not result in full-term pregnancies; furthermore, gene expression profiles
of the iPS cells showed a distinctly different gene expression signature compared to that of
ESCs. In addition, reprogramming was determined to be incomplete, as the epigenetic state
of the iPS cells appeared to lie between that observed in somatic cells and ESCs [60]. In 2007,
Jaenisch's group refined the reprogramming technique by infecting fibroblasts with retroviral
vectors and selecting for the activation of endogenous Oct4 or Nanog genes [61]. This study
showed that DNA methylation, gene expression profiles, and the chromatin state of the
reprogrammed cells resembled embryonic stem cells [61]. Teratomas induced by these cells
contained differentiated cell types representing all three embryonic germ layers; moreover,
the reprogrammed cells from this experiment formed viable chimeras and contributed to the
