The Speed of the Cell Cycle Makes Aging Cells Young Again


When Shinya Yamanaka and his colleagues at the RIKEN Institute discovered a way to reprogram adult cells into embryonic stem cell-like cells, known as induced pluripotent stem cells (iPSCs), they overthrew a core understanding of cell and developmental biology; namely that once cells become committed to a particular cell fate, they irreversibly remain committed to that cell fate.

Most of the work on iPSCs has examined how to increase the efficiency and safety of this reprogramming procedure. The slowness and inefficiency of this process has frustrated stem cell scientists for some time. Even though some progress has been made at increasing the efficiency of the reprogramming process, the “nuts and bolts” of why this procedure is so slow has remained unclear.

However a recent paper from the laboratory of Shangqin Guo at the Yale School of Medicine has revealed a key component of why this procedure is so slow. That component is the speed of the cell cycle or the length of time the cell takes to divide.

Fast-growing cells have lower barriers to keeping the cell committed to a particular cell fate. Thus faster-growing cells are more easily coaxed into being reprogrammed into pluripotency (the ability to differentiate into all adult cell types).

Guo’s research team examined blood cell-forming stem cells in bone marrow. Normally these stem cells are multipotent, which means that they can differentiate into a limited number of adult cell types. The particular type of blood cells that the progeny of these stem cells differentiate into depends on the particular types of growth factors available to the cells.

Guo and others found that these fast growing bone marrow stem cells could be reprogrammed in as little as four cell divisions.  Ultrafast cell cycle is a key feature of these “privileged cells” that can be reprogrammed to efficiently.  Slower-growing stem cells could not be reprogrammed nearly as fast. Thus the length of the cell cycle seemed to be the key to the speed with which cells could be reprogrammed to iPSCs.

This study also has implications for several other applications, besides making individualized iPSCs for patients. Several human diseases are associated with abnormalities in the establishment of proper cell fates and abnormalities in the cell cycle. Therefore, Guo’s paper could provide insights into why certain genetic diseases affect cells the way they do.

Artificial Skin Created Using Umbilical Cord Stem Cells


Major burn patients usually must wait weeks for artificial skin to be grown in the laboratory to replace their damaged skin, buy a Spanish laboratory has developed new protocols and techniques that accelerate the growth of artificial skin from umbilical cord stem cells. Such laboratory-grown skin can be frozen and stored in tissue banks and used when needed.

Growing skin in the laboratory requires the acquisition of keratinocytes, those cells that compose the skin and the mucosal covering inside our mouths.  Keratinocytes can be cultured in the laboratory, but they have a long cell cycle, which means that they take a really long time to divide.  Consequently, cell cultures of keratinocytes tend to take a very long time to grow.

Keratinocytes in culture
Keratinocytes in culture

As they grow, the keratinocytes respond to connective tissue underneath them to receive the cues that tell them how to connect with each other and form either skin or oral mucosa.  In patients with severe burns, however, the underlying connective tissue is also often damaged.  Therefore, finding a way to not only accelerate the growth of cultured keratinocytes, but also to provide the underlying structure that directs the cells to form a proper epithelium is essential.

Remember that severe burn patients are living on borrowed time.  Without a proper skin covering, water loss is severe and dehydration is a genuine threat.  Also, infection is another looming threat.  Therefore, the treatment of a burn patient is a race against time.

Because umbilical cord stem cells grow quickly and effectively in culture, they might be able to differentiate into keratinocytes and form the structures associated with oral mucosa and skin.

University of Granada researchers used a new type of epithelial covering to grow their artificial skin in addition to a biomaterial made of fibrin (the stiff, cable-like protein that forms clots) and agarose to provide the underlying connective tissue. In case you might need a refresher, an epithelium refers to a layer of cells that have distinct connects with each other and form a discrete layer. Epithelia can form single or multiple layers and can be composed of long, skinny cells, short, flat cells, or boxy cells.  An epithelium is a membrane-like tissue composed of one or more layers of cells separated by very little intervening substances.  Epithelia cover most internal and external surfaces of the body and its organs.

Previous work from this same research group showed that stem cells from Wharton’s jelly (connective tissue within the umbilical cord), could be converted into epithelial cells. This current study confirms and extends this previous work and applies it to growing skin, and oral mucosa.

“Creating this new type of skin suing stem cells, which can be stored in tissue banks, mains that it can be used instantly when injuries are caused, and which would bring the application of artificial skin forward many weeks,” said Antonio Campos, professor of histology and one of the authors of this study.

By growing the Wharton’s jelly stem cells on their engineered matrix in a three-dimensional culture system, Campos and his colleagues saw that the stem cells stratified (formed layers), and expressed a bunch of genes that are peculiar to skin and other types of epithelia that cover surfaces (e.g., cytokeratins 1, 4, 8, and 13; plakoglobin, filaggrin, and involucrin).  When examined with an electron microscope, the cells had truly formed the kinds of tight connections and junctions that are so common to skin epithelia.

Electron microscopy analysis of controls and three-dimensional bioactive models of H-hOM and H-hS. SEM images (top) corresponding to N-hOM and N-hS controls showed a tight superficial layer of flat polygonal cells with desquamation signs in which cells were covering the entire surface, whereas samples kept in vitro for 2 weeks showed immature differentiation patterns, and samples implanted in vivo for 40 days tended to resemble the structure of the native control tissues, with flattened cells and evident signs of desquamation. Scale bars = 50 μm. TEM samples (bottom) were analyzed after 40 days of in vivo implantation and demonstrated that in vivo-implanted tissues were mature and well-differentiated, with numerous intercellular junctions, abundant cell organelles, and a collagen-rich stroma. Scale bars = 1 μm. Abbreviations: H-hOM, heterotypical human oral mucosa; H-hS, heterotypical human skin; N-hOM, native human oral mucosa; N-hS, native human skin; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
Electron microscopy analysis of controls and three-dimensional bioactive models of H-hOM and H-hS. SEM images (top) corresponding to N-hOM and N-hS controls showed a tight superficial layer of flat polygonal cells with desquamation signs in which cells were covering the entire surface, whereas samples kept in vitro for 2 weeks showed immature differentiation patterns, and samples implanted in vivo for 40 days tended to resemble the structure of the native control tissues, with flattened cells and evident signs of desquamation. Scale bars = 50 μm. TEM samples (bottom) were analyzed after 40 days of in vivo implantation and demonstrated that in vivo-implanted tissues were mature and well-differentiated, with numerous intercellular junctions, abundant cell organelles, and a collagen-rich stroma. Scale bars = 1 μm. Abbreviations: H-hOM, heterotypical human oral mucosa; H-hS, heterotypical human skin; N-hOM, native human oral mucosa; N-hS, native human skin; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

The authors conclude the article with this statement: “All these findings support the idea that HWJSCs could be useful for the development of human skin and oral mucosa tissues for clinical use in patients with large skin and oral mucosa injuries.”  Think of it folks – new skin for burn patients, quickly, safely and ethically.

Now back to reality – this is exciting, but it is a a pre-clinical study.  Larger animals studies must show the efficacy and safety of this protocol before human trials can be considered, but you must admit that it looks exciting; and without killing any embryos.

See I. Garzón, et al., Stem Cells Trans MedAugust 2013 vol. 2 no. 8625-632.

Genes that prevent iPSC formation


In order to make induce pluripotent stem cells, scientists need to reprogram existing cells to form a cell that is undifferentiated and ready to become whatever we want it to become. Reprogramming is achieved by increasing the concentration of four different proteins within the cells. To increase the intracellular concentration of these proteins, scientists use viruses or other vehicles to import the genes that encode these proteins into the cells and the increased production of these proteins drives cells to become embryonic-like cells that can become anything we want them to be.  Unfortunately, reprogramming, at present, is rather inefficient.

Recently, work from five different labs has shown that two biochemical pathways, the p16INK4A and ARF–p53 pathways, put the brakes on iPSC formation. Five papers in a recent edition of the journal Nature show that the components of this pathway are silenced in iPSCs and strongly expressed in terminally differentiated cells. There are three genes found at the site known as Ink4/Arf (p16Ink4a, p19Arf and p15Ink4b), and these genes are absent in several different types of cancers. For example p16Ink4a is inactive in about 90% of all pancreatic cancers. p15Ink4b is absent from several different blood-based cancers and loss of p19Arf is involved in melanoma formation.  Each one of these gene products acts as a barrier to reprogramming and iPSC formation.

p16INK4A and p19ARF positively regulate the p53 pathway.  p53 inhibits cell proliferation and promotes cellular senescence.  During senescence, the cell essentially takes a nap.  If a you want a cell to grow and participate in healing, the induction of senescence is not a good thing, but if a cell is growing uncontrollably and contributing to a tumor, then forcing a cell into senescence is a good thing.  A protein called p21 (CDKN1A) is also upregulated by p53, and this protein inhibits proliferation and promotes senescence.  Thus p53 is one of the major switches that prevents cell proliferation and promotes senescence.  In 2008, Zhao and coworkers showed that interfering with p53 activity promoted iPSC formation (Zhao, et al., Cell Stem Cell 3, 475-79, 2008).

Hong et al. showed that the absence of, or a reduction in, p53 increases the efficiency of iPS cell generation from mouse and human fibroblasts (Hong, H. et al. Nature 460, 1132–1135 (2009)). Up to 10% of mouse fibroblasts that transiently lacked functional p53 became iPSCs.  Kawamura et al. also enhanced the reprogramming of mouse embryonic fibroblasts (MEFs) by reducing the level of p21 or ARF (Kawamura, T. et al. reprogramming. Nature 460, 1140–1144 (2009)). Since ARF inhibits p53 degradation, ARF knockdown might enhance reprogramming by decreasing p53 stability.  All of these studies, using various approaches, showed that p53 depletion enhances iPS cell generation.

Li et al. and Utikal et al. observed that the INK4/ARF locus is silenced in iPS cells reprogrammed from Mouse Embryonic Fibroblasts (MEFs), as well as in embryonic stem cells, but not in MEFs (Li, H. et al. Nature 460, 1136–1139 (2009) & Utikal, J. et al. Nature 460, 1145–1148 (2009)).  Utikal et al. also observed that older MEFs, which harbor increased levels of p16INK4A, ARF and p21 owing to ageing and the onset of senescence, show a decrease in reprogramming efficiency. Li et al. also linked ageing and expression from the INK4/ARF locus with decreased iPS cell generation. They showed that cells from old mice express genes at this locus at a higher level than cells from young mice and that this is associated with a decreased reprogramming efficiency, which can be rescued by knocking down INK4/ARF.

As a final caveat, Marión et al. found that p53 prevents the reprogramming of MEFs that have various types of DNA damage (Marión, R. M. et al. Nature 460, 1149–1153 (2009)). Although loss of p53 function allows faster and more efficient reprogramming in the presence of DNA damage, it generates iPS cells that contain damaged DNA and chromosomal abnormalities. This emphasizes that, although these studies provide crucial mechanistic insight into how the generation of iPS cells is regulated, it will be important to determine how the p16INK4A and ARF–p53 tumor suppressor pathways can be silenced to allow the efficient production of iPS cells without increasing the possibility of making cell lines that contain mutations that predispose them to malignant tumor formation.

Surely iPSC production represents the future of regenerative medicine.  We need not kill human embryos, and the time required to make iPSCs can be substantially cut with this technology as it is honed and even made safer.