Rejuvenation Factor Discovered in Human Eggs


When the egg is fertilized by a sperm, it is transformed into a single-celled embryo or zygote that is metabolically active and driven to divide and develop. The egg, on the other hand, is a rather inert cell from a metabolic perspective. What is it in the egg that allows it to transform into something so remarkably different?

A new study by Swea-Ling Khaw and others in the laboratory of Ng Shyh-Chang at the Genome Institute of Singapore (GIS) has elucidated two main factors that help rejuvenate the egg and might also help reprogram adult cells into induced pluripotent stem cells (iPSCs).

Eggs express large amounts of a protein called Tcl1. Tcl1 suppresses the function of old, potentially malfunctioning mitochondria (the structure in cells that makes the energy for the cell). This suppression prevents damaged mitochondria from adversely affecting the egg’s transformation from into an embryo.

Remember also that if an adult cell is fused to an egg, it can cause the egg to divide and form an early embryo. Therefore, the egg cytoplasm is able to reprogram adult cells as well, and Tcl1 seems to play a role in this reprogramming capability as well.

In a screen for genes that are important to the reprogramming process, Shyh-Chang’s laboratory isolated two genes, Tcl1 and Tcl1b1. Further investigation of these two proteins showed that Tcl1 affects mitochondria by inhibiting a mitochondrial protein called polynucleotide phosphorylase (PNP). By locking PNP in the cytoplasm rather than the mitochondria, the growth and function of the mitochondria are inhibited. Tcl1b1 activates the Akt kinase, which stimulate cell growth, survival, and metabolism.

In a review article in the journal Stem Cells and Development, Anaïs Wanet and others explain that energy production in pluripotent stem cells is largely by means of glycolysis, which occurs in the cytoplasm. Mitochondria in pluripotent stem cells are immature subfunctional. When adult cells are reprogrammed into iPSCs, mitochondria function is shut down and energy production is largely derived from glycolysis. When the cells differentiate, the mitochondria are remodeled and become functional once again. Tcl1 is the protein that help shut down the mitochondria so that the pluripotent state can ensue and Tcf1b1 gears up the pluripotent stem cells to grow and divide at will.

Given this remarkable finding, can Tcf1 help make better iPSCs? Almost certainly, but how does one use this important factor to make better iPSCs?  That awaits further experimentation.  Additionally, this finding might also help aging and infertility issues as well. Hopefully this work by Shyh-Chang and her colleagues will lead to many more fruitful and exciting experiments.

Differential Immunogenicity of Cells Derived from Induced Pluripotent Stem Cells


Induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific pluripotent stem cells may become a renewable source of a patient’s own cells for regenerative therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood.

Using a humanized mouse model (denoted Hu-mice) with a functional human immune system, Yang Xu and his colleagues from UC San Diego has shown that most teratomas or tumors formed by human iPSCs were readily recognized by immune cells and rejected. However, when these human iPSCs were differentiated into smooth muscle cells or retinal pigmented epithelial cells, the results were rather different. Human iPSC-derived smooth muscle cells appear to be highly immunogenic, but human iPSC-derived retinal pigment epithelial (RPE) cells are tolerated by the immune system, even when transplanted outside the eye.

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When Xu and others examined these results more closely, they discovered that this differential immunogenicity is due to the abnormal expression of cell surface proteins in hiPSC-derived Smooth Muscle Cells, but not in hiPSC-derived RPEs.

These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration. They also show that iPSC lines must be individually screened to determine if their derivatives are recognized by the patient’s immune system as foreign.

These results were published in Cell Stem Cell.

Reprogramming Pluripotent Cells into Totipotent Cells


With the advent of the Nobel Prize-winning research of Shinya Yamanaka, scientists are presently able to reprogram mature, adult cells into pluripotent cells. These “induced pluripotent stem cells” or iPSCs are made by genetically engineering adult cells and culturing them in special conditions. These iPSCs can also be differentiated, potentially, into every cell type in the adult human body. Now Maria-Elena Torres-Padilla‘s research team is trying to push the limits of stem cell science even further.

Torres-Padilla and her coworker successfully made “totipotent” cells, which have the same characteristics as those of the earliest embryonic stages, from pluripotent stem cells. This work was the result of a fruitful collaboration between Juanma Vaquerizas from the Max Planck Institute for Molecular Biomedicine (Münster, Germany), and Maria-Elena Torres-Padilla and her colleagues at the Institute of Genetics and Molecular and Cellular Biology (IGBMC) at Illkirch, France. This work has been published in the journal Nature Structural & Molecular Biology.

Soon after fertilization, the embryo begins it first rounds of cell division, which is known as the “early cleavage” stages. At this early stage of development, when the embryo is composed of only 1 or 2 cells, the “blastomeres” or cells of the embryo are “totipotent.” Totipotent means that these blastomeres can produce an entire embryo, or the placenta and umbilical cord that accompany it.

Early Cleavage Stages

After the 12-16-cell stage, the embryo undergoes a complex process called “compaction,” in which the cells become very tightly bound together and two populations of cells become apparent.

Post-Compaction Embryo

Post-Compaction Embryo

The cells on the inside, which give rise to the cells of the “inner cell mass” (ICM), and outside cells, which give rise to the “trophectoderm” that produce the placenta. Trophectoderm cells express a specific set of genes; Yap1, Tead4, Gata3, Cdx2, Eomes and Elf5. The ICM generates the embryo proper and the ICM cells express a cadre of genes specific to these cells; Oct4, FGF4, Sall4, Sox2 and Nanog. These ICM cells are no longer totipotent, but have become “pluripotent,” can differentiate into any tissue, but they cannot alone produce the placenta or a whole embryo. As development progresses, these pluripotent cells continue to specialize and form the various tissues of the body through the process of cellular differentiation.

While it is possible to make pluripotent cells from mature, adult cells, Torres-Padilla and her coworkers have studied the characteristics of totipotent cells of the embryo and discovered factors capable of inducing a totipotent-like state.

When they cultured pluripotent stem cells in culture, a small percentage of totipotent cells appear spontaneously. Such cells are called “2C-like cells,” after their resemblance to the 2-cell stage embryo. Torres-Padilla and her team compared these 2C-like cells to totipotent cells in early embryos in order to determine their common characteristics and the features that distinguish them from pluripotent cells. In particular, Torres-Padilla and her collaborators found that the chromosomes of totipotent cells were less condensed and that the amount of the protein complex CAF1 was diminished. CAF1 (Chromatin Assembly Factor 1) is a protein complex that helps assemble histone proteins onto DNA.

CAF1

Histones act as tiny spools around which DNA is wound. Because DNA is negatively charged, and histones are positively charged, the two have a natural affinity for each other. CAF1 binds to histones and regulates the association of histones with DNA in order to ensure that the assembly of histones on DNA is and orderly process. Histones wind DNA into a tight structure called “chromatin.” CAF1, as it turns out, is responsible for maintaining the pluripotent state by ensuring that the DNA remains properly wound around histones.

chromatin+structure

As an extension of this hypothesis, Torres-Padilla and her crew were able to induce a totipotent state by inactivating the expression of the CAF1 complex. CAF1 inactivation caused the chromatin of pluripotent cells to reform into a less condensed state, and this less condensed state was conducive to totipotency.

These data provide new avenues for understanding the nature of pluripotency, and could increase the efficiency of reprogramming somatic cells to be used for applications in regenerative medicine,

New York Stem Cell Foundation Invents Robotic Platform for Making Induced Pluripotent Stem Cells


Induced pluripotent stem cells (iPSCs) are made from mature adult cells through a combination of genetic engineering and cell culture techniques. Because they are made from cells isolated from specific patients, they are patient-specific cells that can be used for drug testing, model experimental systems, and potentially cells for regenerative therapies.

Induced-pluripotent-stem-cell-picture.jpg

Unfortunately, iPSCs are made in different laboratories that use different reagents and different protocols and with workers with different skill levels. Consequently, laboratory-made iPSC lines show a very wide range of variation that are not due to genetic differences in the cells from which they were made. Additionally, the production of iPSCs is labor intensive and expensive and there is a deep need to standardize the whole process. What are stem-cell scientists to do?

New York Stem Cell Foundation (NYSCF) has announced the development of a robot-driven apparatus that automates and completely standardizes the production of iPSCs. This modular, robotic platform for iPSC reprogramming enables automated, high-throughput conversion of cells isolated from skin biopsies into iPSCs and differentiated cells derived from them with minimal manual intervention. In a paper in the journal Nature Methods, NYSCF scientists in collaboration with bioengineers demonstrates that automated reprogramming of mature cells with this robotic platform (that uses pooled selection of pluripotent cells) results in high-quality, stable iPSC lines. These lines show less line-to-line variation than either manually produced iPSC lines or iPSC lines produced through automation followed by single-colony subcloning.

“The capacity to test drugs on thousands of patients in a dish will change how we cure disease. We
will be more informed about how drug candidates will behave in patients before the clinical trial phase accelerating the discovery process. This technology will enable us to bring precision medicine
treatments and personalized pharmaceuticals to more patients,”noted Dr. Thomas Singer, Senior
Vice President, F. Hoffmann-La Roche Ltd, Pharmaceuticals Division.
“This technology may help us predict how drug candidates behave in patients before the most complex and expensive phase of drug development: clinical trials. This insight could speed up new biomedical R&D and open the door to a larger number of high impact precision therapies,”said Freda Lewis-Hall, M.D., DFAPA, Chief Medical Officer and Executive Vice President, Pfizer Inc.
“Our goal is to understand and treat diseases. This is not an artisanal pursuit. Researchers need to
look at genetically diverse populations at scale, which means creating large numbers of standardized, human pluripotent stem cells. The NYSCF Global Stem Cell Array’s massive parallel processing
capabilities make this research possible,” said NYSCF Research Institute CEO and Founder Susan
L. Solomon, an author of the paper.

This robotic platform can potentially enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized clinical treatments.

Drug Treatment Enhances Regeneration in Adult Mice


Humans do not have the ability to regenerate lost limbs, but there are particular vertebrate animals that possess this remarkable ability. Amphibians, for example, can regenerate lost appendages and this ability has made these remarkable creatures the focus of a good deal of research in order to understand how to translate this regenerative ability into other mammals, such as ourselves.

Mammals, unfortunately, have no such regenerative ability and we also tend to form scars over injury sites as a result of wound repair. Fortunately, there are some experimental mammalian models that do display enhanced wound regeneration and these organisms permit the study of the underlying processes at play during regeneration. Many of the processes which mediate wound regeneration are controlled through regulatory mechanisms stimulated by a protein called the “hypoxia-inducible factor-1a” or HIF-1α transcription factor. The laboratory of Ellen Heber-Katz at the Wistar Institute in Philadelphia, PA, has worked hard to characterize the function and specific biological activity of HIF-1α in MRL (Murphy Roths Large) mice. MRL mice show spontaneous regenerative healing. In a new study, Heber-Katz and her colleagues confirmed the importance of HIF-1α in regeneration and extended their understanding of this protein by establishing that small molecule-mediated stabilization of HIF-1α protein in wounded animals promotes regenerative wound healing after injury in mouse strains that do not possess the ability to spontaneously regenerate.

HIF-1alpha activity

Initial studies wounding healing in mice that had been subjected to ear-hole punch injuries. Such injuries induced high HIF-1α levels only in MRL mice but not in control animals (C57BL/6 mice). Also, if small interfering RNAs were used to knock down the levels of HIF-1α in MRL mice, wound closure was delayed and inefficient. These data suggested that HIF-1α was essential for spontaneous healing, and implied that artificial induction of HIF-1α at injury sites in strains of mice that do not show spontaneous healing could induce regenerative healing.

Heber-Katz and her co-workers subjected Swiss Webster mice (a mouse strain that does not show spontaneous regeneration) to ear-punch injuries, but also treated their ear injuries with a hydrogel laced with a drug called 1,4-DPCA (1,4-dihydrophenonthrolin-4-one-3-carboxylic acid). 1,4-DPCA is an inhibitor of the prolyl hydroxylases (PHDs) enzymes that degrade HIF-1α. This did not work because the mice kept grooming their ears and wiping off the hydrogel. So, an undaunted Heber-Katz team implanted the hydrogel underneath the skin of the animals’ necks. They observed HIF-1α expression increase on day 1 and reached maximal expression on days 3 to 4 after injection. If they repeated injection of 1,4-DPCA every 5 days in the neck, the full wound closed on day 35, with no harmful long-term effects observed at 3 months post-injection.

How does HIF-1α enable regenerative wound healing in these mice? Interestingly, 1,4-DPCA treatment induced the expression of stem-cell marker genes such as Nanog and Sox2. These induced cellular de-differentiation in the cells and stimulated the very early and rapid re-casting of the cell layers over the wound that are so characteristic of regeneration, but not wound repair. Additionally, 1,4-DPCA treatment reduced tissue remodeling, inflammatory responses, and scar formation. It also stimulated promoted events associated with the latter stages of the regenerative processes; such as the growth of new cells and the growth and redifferentiation of those cells into things like cartilage, and hair.

These data suggest that induction of the transcription factor HIF-1α promotes tissue regeneration over scar formation in mammals. Thus this is a potential strategy to stimulate the regeneration of lost or damaged tissues. The non-toxic and specific nature of the HIF-1α inhibitor used in these experiments should lead to fruitful studies in appropriate human systems (cell culture) but although mouse models and maybe larger animal models as well will assess the ability of particular drugs to induce repair in other parts of the body and should prove equally as useful.

This new study was published in Science Translational Medicine.

Celprogen Markets a Xeno-Free Medium for Stem Cells


Celprogen Inc., has designed a cell culture medium for growing stem cells of all types in the laboratory that does not contain any animal products. Such a medium is called “Xeno-free.”

This new xeno-free medium, XFS2 can successfully culture induced pluripotent stem cells, primary human cells, stem cells and progenitor cells. XFS2 can also be used to grow cancer cell for research purposes.

Growing stem cells in cell culture requires a unique mixture of growth factors that stimulate cell proliferation and cell survival. Because stem cells can grow in XFS2 without differentiating, it can be used to grow cells for clinical trials s well.

Celprogen scientists have used XFS2 to grow human embryonic stem cells, fat-derived stem cells, and stem cells isolated from specific human organs. A chief advantage of XFS2 is that it does not contain any human serum, which is essential for clinical applications, since patients could have serious immunological reactions against serum that does not come from their own blood.

When stem cells grown in XFS2 were compared with stem cells grown in other cell culture media, the XFS2-grown cells did not display any detectable differences from their serum-grown counterparts. Furthermore, according to Celprogen, stem and progenitor cells grew robustly in XFS2. We will certainly need to see the reactions of laboratories who chose to use XFS2 to grow their stem cells before we can confirm or deny this.

Celprogen hopes that their safe and xeno-free XFS2 medium will facilitate the potential application of stem cell transplantation for the treatment of various diseases, and Celprogen is equally hopeful that their new medium will prove itself to be safe, give reproducible results, and a high-quality medium for the propagation of stem and progenitor cells in the laboratory.

Has anyone used XFS2 to grow their stem or progenitor cells in the laboratory yet?  If so, let me know how it works.

Adding Cyclosporin to Bone Marrow Might Increase Stem Cell Numbers, Quality, and Engraftment Efficiency


In the bone marrow, we have an army of blood cell-making stem cells called hematopoietic stem cells (HSCs) that make all the blood cells that course through our blood vessels. These cells divide throughout our lifetimes, and they replacement themselves while they generate all the red and white cells found in our blood.

hematopoietic-stem-jpg

HSCs are also the cells that are harvested during bone marrow aspirations and biopsies. Transplantation of HSCs can save the lives of patients with blood cancers or other types of blood-or bone marrow-based diseased.

Harvesting and transplanting HSCs is, therefore, a very important clinical strategy for treating many different types of blood disorders and diseases. However, this crucial strategy is limited by the relative rarity of HSCs in isolated bone marrow. Additionally, the number and function of HSCs deteriorate both during their collection from the bone marrow (BM) and during their manipulation outside the body. Fortunately, the development of culture conditions that best mimic the environment these cells experience in bone marrow (the so-called “HSC niche environment”) may help to minimize this loss.

Scanning electron microscopy of stem cells (yellow / green) in a scaffold structure (blue) serving as a basis for the artificial bone marrow.

Scanning electron microscopy of stem cells (yellow / green) in a scaffold structure (blue) serving as a basis for the artificial bone marrow.

One of the most important variables for HSC viability is oxygen concentration, since various studies have shown that the oxygen concentrations found in ambient air seems to be damaging to HSCs, which normally are found in rather oxygen-poor reaches in bone marrow. Researchers from the laboratory of Hal Broxmeyer at the Indiana University School of Medicine have discovered that HSCs suffer from ‘‘extra-physiologic oxygen shock/stress (EPHOSS)” if they are harvested under ambient oxygen conditions. On top of that, treatment of the collected HSCs with the immunosuppressant drug cyclosporin A (CSA) can inhibit this stress, enhance the yield of collected HSCs, and increase their transplantation efficiency.

When Broxmeyer and his colleagues compared mouse BM that had been harvested under normal oxygen concentrations (21% O2) and low-oxygen concentrations (3% O2), they observed that the hypoxic (low-oxygen) treatment caused a 5-fold increase in the number of Long Term (LT) self-renewing HSCs, and a decrease in harmful reactive oxygen species (ROS) and mitochondrial activity. Broxmeyer and others also confirmed the positive effect of hypoxia on HSC collection from human cord blood. When mouse BM collected under different conditions were assayed by competitive transplantation, the “hypoxic HSCs” engrafted more efficiently in recipient mice. This increased engraftment was not due to enhanced homing or reduced cell death. Instead it seems that the stress response to non-physiological oxygen concentrations (EPHOSS) has a rapid and significant damaging effect in HSCs.

Broxmeyer decided to take this study one step further. In mitochondria (the powerhouse of the cell), increased expression of the mitochondrial permeability transition pore (MPTP) seems to be one of the key mechanism by which oxidative stress affects HSCs.

mitochondrial permeability transition pore

mitochondrial permeability transition pore

Induction of the MPTP leads to mitochondrial swelling and uncoupled energy production (which leads to the generation of reactive oxygen species, otherwise known as “free radicals). This leads to cell death apoptosis and necrosis, and intermittent MPTP activation may also decrease stem cell function in general without killing the cells. Broxmeyer and his coworkers came upon a rather ingenious idea to use the drug cyclosporin A (CSA) to antagonize MPTP induction, since CSA inhibits the associated CypD (cyclophilin) protein. When HSCs were collected under high-oxygen conditions in the presence of CSA, there was a 4-fold increase in the recovery of LT-HSCs and enhanced engraftment levels compared to HSCs harvested in high-oxygen conditions without CSA. This link was further strengthened by examining the HSCs of mice with a deletion of the CypD gene. In these mice, HSCs collected under high-oxygen conditions showed increased LT-HSC recovery and decreased LT-HSC ROS levels compared to wild-type mice.

Cyclophilin

Cyclophilin

How, harvesting and processing HSCs from bone marrow in a low-oxygen environment within a transplant clinic is generally not possible. However, given the observed advantages, the application of CSA may represent an easy and attractive alternative. The authors of this paper (which was published in the journal Cell) note that CSA is already used in the clinic as an immunosuppressant. Therefore, this technique could potentially be rapidly adapted into bone marrow harvesting techniques.

An additional thought is that studies that use other types of stem cells for transplantation might also need to consider the effects of EPHOSS and oxygen concentration while preparing their cells in other model systems.

See “Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock” from Cell by Stuart P. Atkinson