Gum Nerve Cells Become Tooth-Specific Mesenchymal Stem Cells

Stem cells self-renew and also produce progeny that differentiate into more mature cell types. The neurons and glia that compose nervous systems are examples of mature cells and these cells can be produced from embryonic stem cells, induced pluripotent stem cells, or neural stem cells. However, the reverse does not occur during development; more mature cells do not de-differentiate into less mature cells types. Development tends to be a one-direction event.

However, researchers have now discovered that inside teeth, nervous system cells can transform back into stem cells. This unexpected source of stem cells potentially offers stem cell scientists a new starting point from which to grow human tissues for therapeutic or research purposes without using embryos.

“More than just applications within dentistry, this finding can have very broad implications,” says developmental biologist Igor Adameyko of the Karolinska Institute in Stockholm, who led this new work. “These stem cells could be used for regenerating cartilage and bone as well.”

The soft “tooth pulp” in the center of teeth has been known to contain a small population of tooth-specific mesenchymal stem cells, which can typically differentiate into tooth-specific structures, bones, and cartilage. However, no one has conclusively determined where these stem cells came from. Adameyko hypothesized that if he could trace their developmental lineage, he should be able to recapitulate their development in the laboratory. This might offer new ways of growing stem cells for tissue regeneration.

Adameyko and his and colleagues had already studied glial cells, which are nervous system cells that surround neurons and support them. Several of the nerves that wind through the mouth and gums help transmit pain signals from the teeth to the brain are associated with glial cells.

Adameyko and others used fluorescent labels to mark the glial cells in the gum. When the gum-specific glial cells were observed over time, some of these cells migrated away from neurons in the gums into teeth, where they differentiated into mesenchymal stem cells. These same cells then matured into tooth cells. This work was reported in the journal Nature.

a–c, Incisor traced for 3 days from adult PLP-CreERT2/R26YFP mouse. Note protein gene product 9.5 (PGP9.5)+ nerve fibres (a). b, c, Magnified areas from a. d, e, Incisor traced for 30 days from adult PLP-CreERT2/R26YFP mouse. Note collagen IV+ blood vessels (d). e, YFP+ odontoblasts and adjacent pulp cells. f, Incisor traced for 30 days from Sox10-CreERT2/R26YFP mouse. g–k, Incisor traced for 40 days from PLP-CreERT2/R26Confetti incisor. h–j, Magnified areas from g. Arrow in h indicates a cluster of odontoblasts; arrow in j points at CFP+ and RFP+ cells in proximity to a cervical loop at the base of CFP+ and RFP+ streams shown in g and i. k, Streams of CFP+ and RFP+ pulp cells next to i and j. l, m, Incisor traced for 40 days from PLP-CreERT2/R26Confetti mouse with YFP+ and RFP+ pulp cells adjacent to clusters of odontoblasts with corresponding colours. m, Magnified region from l. n, Stream of pulp cells (arrows) in proximity to the cervical loop; yellow and red isosurfaces mark YFP+ and RFP+ cells. o, p, Progenies of individual MSCs intermingle with neighbouring clones in pulp (o) and odontoblast layer (p), projections of confocal stacks. q, r, Clonal organization of mesenchymal compartment in adult incisor. a–n, Dotted line, enamel organ and mineralized matrix. Scale bars, 100 µm (a, d, f, g, k, l); 50 µm (b, c, e, m–p). CL1 and CL2 indicate labial and lingual aspects of cervical loop. d.p.i., days post-injection. s, Incidence of mesenchymal clones depending on fraction of odontoblasts within the clone. t–v, Proximity of dental MSCs (dMSCs) to cervical loop (CL) correlates with clonal size and proportion of odontoblasts in clone.

Before this experiment, it was generally believed that nervous system cells were unable to de-differentiate or revert back to a flexible stem cell state. Therefore, Adameyko said that it was very surprising to see such a process in action. He continued: “Many people in the community were convinced … that one cell type couldn’t switch to the other. But what we found is that the glial cells still very much maintain the capacity” to become stem cells. If stem cell researchers and physicians could master those chemical cues in the teeth pulp that signals glial cells to transform into mesenchymal stem cells, they could generate a new way to grow and make stem cells in the lab.

“This is really exciting because it contradicts what the field had thought in terms of the origin of mesenchymal stem cells,” says developmental biologist Ophir Klein of the University of California, San Francisco, who was not involved in the new work. But it’s also just the first step in understanding the interplay between the different cell populations in the body, he adds. “Before we really put the nail in the coffin in terms of where mesenchymal stem cells are from, it’s important to confirm these findings with other techniques.” If that confirmation comes, a new source of stem cells for researchers will be invaluable, he says.

Identifying Barriers to Cell Reprogramming

A new study from the laboratory of Miguel Ramalho-Santos, associate professor of obstetrics, gynecology and reproductive sciences at the University of California, San Francisco (UCSF), might lead to a faster way to derive stem cells that can be used for regenerative therapies.

Induced pluripotent stem cells or iPSCs, which are made from adult cells by means of genetic engineering and cell culture techniques, behave much like embryonic stem cells. These adult cell-derived stem cells are pluripotent and can be differentiated into heart, liver, nerve and muscle cells. This present work by Ramalho-Santos and his colleagues builds upon the reprogramming protocols that have been developed to de-differentiate mature adults cells into iPSCs.

Ramalho-Santos and his co-workers have been interested in understanding the reprogramming process more completely in order to increase the efficiency and safety of this process. In particular, the Ramalho-Santos laboratory has been examining the cellular barriers that prevent adult cells from being reprogrammed in order to circumvent them and increase the efficiency of stem-cell production. In this present work, Ramalho-Santos’ group identified many of these cellular barriers to reprogramming.

“Our new work has important implications for both regenerative medicine and cancer research,” said Ramalho-Santos, who is also a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

In 2012, Shinya Yamanaka from Kyoto University won the Nobel Prize in Physiology or Medicine for his discovery of iPSCs. Yamanaka discovered ways to turn back the clock on adult cells, but the protocol that he developed and others have used for years is inefficient, slow, and tedious. The percentage of adult cells successfully converted to iPS cells is usually rather low, and the resultant cells often retain traces of their earlier lives as mature, fully-differentiated cells.

To make iPSCs, researchers force the expression of pluripotency-inducing genes in adult cells. These four genes (Oct4, Klf4, Sox2, cMyc) have become known as the so-called “Yamanaka factors” and they work to turn back the clock on cellular maturation. However, as Ramalho-Santos explained: “From the time of the discovery of iPS cells, it was appreciated that the specialized cells from which they are derived are not a blank slate. They express their own genes that may resist or counter reprogramming.”

So what are those barriers? Ramalho-Santos continued: “Now, by genetically removing multiple barriers to reprogramming, we have found that the efficiency of generation of iPS cells can be greatly increased.” This discovery will contribute to accelerating the production of safe and efficient iPSCs and other types of other reprogrammed cells, according to Ramalho-Santos.

Instead of identifying individual genes that act as barriers to reprogramming, Ramalho-Santos and others discovered that sets of genes acted in combination to establish barriers to reprogramming. “At practically every level of a cell’s functions there are genes that act in an intricately coordinated fashion to antagonize reprogramming,” Ramalho-Santos explained. These existing mechanisms probably help mature, adult cells maintain their identities and functional roles. Ramalho-Santos explained it this way: “Much like the Red Queen running constantly to remain in the same place in Lewis Carroll’s ‘Through the Looking-Glass,’ adult cells appear to put a lot of effort into remaining in the same state.” Ramalho-Santos also added that apart from maintaining the integrity of our adult tissues, the barrier genes probably serve important roles in other diseases, including in the prevention of certain cancers

To identify these barriers, Ramalho-Santos and his team had to employ cutting-edge genetic, cellular and bioinformatics technologies. They collaborated with other UCSF labs headed by Jun Song, assistant professor of epidemiology and biostatistics, and Michael McManus, associate professor of microbiology and immunology.

They conducted genome-wide RNAi screens that revealed known and novel barriers to human cell reprogramming. Of these, a protein called ADAM29 antagonizes reprogramming as does clathrin-mediated endocytosis, which antagonizes reprogramming by enhancing TGF-β signaling. Also it became apparent that different barrier pathways have a combined effect on reprogramming efficiency. Additionally, genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion also act as barriers to reprogramming.

The hopes are that this knowledge will produce iPSCs faster that are safer to use and differentiate more completely.

Gene Inhibitor Plus Fish Fibrin Restore Nerve Function Lost After a Spinal Cord Injury

Scientists at UC Irvine’s Reeve-Irvine Research Center have discovered that injections of salmon fibrin injections into the injured spinal cord plus injections of a gene inhibitor into the brain restored voluntary motor function impaired by spinal cord injury.

Gail Lewandowski and Oswald Steward, director of the Reeve-Irvine Research Center at UCI, examined rodents that had received spinal cord injuries.  They were able to heal the damage by developmentally turning back the clock in a molecular pathway that is critical to the formation of the corticospinal nerve tract, and by providing a scaffold for the growing neurons so that the axons of these growing neurons could grow and make the necessary connections with other cells.  Their research was published in the July 23 issue of The Journal of Neuroscience.

The work of Steward and Lewandowski is an extension of previous research at UC Irvine from 2010.  Steward and his colleagues discovered that the axon of neurons grow quite well once an enzyme called PTEN is removed from the cells.  PTEN is short for “phosphatase and tensin homolog,” and it removes phosphate groups from specific proteins and lipids.  In doing so, PTEN signals to cells to stop dividing and it can also direct cells to undergo programmed cell death (a kind of self-destruct program).  PTEN also prevents damaged tissues from regenerating sometimes, because it is a protein that puts the brakes of cell division.  Mutations in PTEN are common in certain cancers, but the down-regulation of PTEN is required for severed axons to re-form, extend, migrate to their original site, and form new connections with their target cells.

 

After two years, team from U.C. Irvine discovered that injections of salmon fibrin into the damaged spinal cord or rats filled cavities at the injury site and provided the axons with a scaffolding upon which they could grow, reconnect and facilitate recovery. Fibrin produced by the blood system when the blood vessels are breached and it is a fibrous, insoluble protein produced by the blood clotting process.  Surgeons even use it as a kind of surgical glue.

“This is a major next step in our effort to identify treatments that restore functional losses suffered by those with spinal cord injury,” said Steward, professor of anatomy & neurobiology and director of the Reeve-Irvine Research Center. “Paralysis and loss of function from spinal cord injury has been considered irreversible, but our discovery points the way toward a potential therapy to induce regeneration of nerve connections.”

In their study, Steward and Lewandowski subjected rats to spinal cord injuries, and then assessed their defects.  Because these were upper back injuries, the rats all showed impaired forelimb (hand) movement.  Steward and Lewandowski then treated these animals with a combination of salmon fibrin at the site of injury and a modified virus that made a molecule that inhibited PTEN.  These viruses were genetically engineered adenovirus-associated viruses encoded a small RNA that inhibited translation of the PTEN gene (AAVshPTEN).  This greatly decreased the levels of PTEN protein in the neurons.  Other rodents received control treatments of only AAVshPTEN and no salmon fibrin.

The results were remarkable.  Those rats that received the PTEN inhibitor alone showed no improvement in their forelimb function, but those animals who were given AAVshPTEN plus the salmon fibrin recovered forelimb use (at least reaching and grasping).

“The data suggest that the combination of PTEN deletion and salmon fibrin injection into the lesion can significantly enhance motor skills by enabling regenerative growth of corticospinal tract axons,” Steward said.

Statistics compiled by the Christopher & Dana Reeve Foundation suggests that approximately 2 percent of Americans have some form of paralysis that is the result of a spinal cord injury.  Spinal cord injuries break connections between nerves and muscles or nerves and other nerves.  Even injuries the size of a grape can cause complete loss of function below the level of the injury.  Injuries to the neck can cause paralysis of the arms and legs, an absence of sensation below the shoulders, bladder and bowel incontinence, sexual dysfunction, and secondary health risks such as susceptibility to urinary tract infections, pressure sores and blood clots due to an inability to move one’s legs.

Steward said the next objective is to learn how long after injury this combination treatment can be effectively administered. “It would be a huge step if it could be delivered in the chronic period weeks and months after an injury, but we need to determine this before we can engage in clinical trials,” he said.

Human Umbilical Cord Mesenchymal Stem Cells Form Prostate Gland Tissues

Repairing the prostate gland is an important goal in regenerative medicine. However, finding the right cell for the job has proven to be a slow and tedious search.

To that end, Wei-Qiang Gao and his colleagues from Shanghai Jiao Tong University in Shanghai, China, used mesenchymal stem cells from human umbilical cord (hUC-MSCs) to test the ability of these cells to differentiate into prostate-specific cells. They combined hUC-MSCs with rat urogenital sinus stromal cells (rUGSSs) and then transplanted these cells into the renal capsule of BLB/c nude mice for two months. Cells tend to grow very well under the kidney capsule because this particular microenvironment has a very rich blood supply. Also the rUGSSs provide soluble, secreted factors that induce the hUC-MSCs to differentiate into prostate-specific cells.

After removing the implanted tissue, analyses of the implanted cells showed that the hUC-MSCs differentiated into prostate epithelial-like cells. This was confirmed by the presence of prostate specific antigen on the surfaces of these hUC-MSCs. Prostate specific antigen is only found on prostate cells, which is the reason why this protein is such a good indicator of prostate cancer. Also, the hUC-MSCs formed prostatic glandular structures that had the same cellular architecture as a normal prostate (see figure F below). Additionally, the human origin of the hUC-MSCs was further confirmed by the detection of a protein called human nuclear antigen, which is specific to human cells.

Human UC-MSCs combined with rUGSSs can generate prostate glands. Mice were sacrificed 2 months after co-transplantation surgery, and the kidneys from the cell implanted nude mice were collected. (A) Graft initiated with hUC-MSCs alone and (B) rUGSSs alone were used as negative control, respectively. (C) Graft derived with hUC-MSCs and rUGSSs. (D–F) Histological analyses of the sections of the graft stained for haematoxylin and eosin (H&E). (D) Note that while hUC-MSCs alone and (E) rUGSSs single cell type transplantation fail to regenerate prostate glandular structures. (F) co-transplantation of hUC-MSCs and rUGSSs gives rise to prostate glandular structures. Scale bar 50 mm.

This interesting paper shows that hUC-MSCs can differentiate into epithelial-like cells that are normally derived from embryonic endodermal tissue. This implies that MSCs from umbilical cord can be used to repair not only prostate glands, but also other endodermally-derived tissues.

Ovarian Surface Stem Cell Population Identified in Mice

Approximately once a month, pre-menopausal women undergo ovulation. During ovulation, a mature graafian follicle is ejected from a rupture in the surface of the ovary. How does the ovary repair itself each month? Many have suspected that the ovary contains its own resident stem cell population that divides and heals the ovary after each ovulation. However, demonstrating the presence of this stem cell population and isolating the ovarian stem cells responsible for this feat have proven difficult.

Now, Nick Barker and colleagues from the University of Edinburgh have identified a cell surface protein called a “Leucine-rich-repeat-containing G protein coupled receptor 5” (LGR5) that is expressed on the surfaces of cells in the ovary surface layer. These LGR5-expressing cells seem to be the stem cell population that contributes to the ovary and oviduct covering during the formation of these organs, and then persist to repair and maintain the ovary surface layer in adults.

LGR5 is also expressed in the cells that cover several other organs. Experiments in mice have shown that LGR5+ cells are in the developing ovary from embryonic day 13.5 onwards. These LGR5-expressing cells are first detected in the ovary surface and lower layers, but later only in the surface of the ovaries of newborn mice. In adult ovaries, LGR5-expressing cells are found only in the layer of cells at the ovary surface, and in the cells that cover the far reaches of the oviduct, and in the cells that line the mesovarium ligament. These LGR5+ cells are found throughout the ovary surface, but they are most prominent in ovulating regions where the ovary surface ruptures. Barker and others also showed that in undamaged regions of the ovary, only a small proportion of dividing cells were LGR5+, but in ovaries that suffered ovulation-induced damage, cycling LGR5+ cells comprised a higher proportion (~50%) of the total percentage of ovary surface cells. These observations do not prove, but are wholly consistent with LGR5-expressing cells playing a seminal role in ovary tissue repair.

Lineage studies with marked cells in embryonic and fetal mice showed that the LGR5+ cells present during ovary development contributed to adult LGR5+ cells that are part of the ovary covering. These lineage studies show that the embryonic and fetal LGR5+ cells are the source of adult stem cells that maintain the ovary surface.

Lineage studies in adult females showed that those cells closer to damaged areas of the ovary underwent more cell divisions. Also, the marked cells on the ovary surface were not only capable of self-renewal, but they could also differentiate into more than one cell type (epithelia of the mesovarian ligament and oviduct/fimbria); this is a key characteristic that defines a stem cell population.

This Barker’s study not only more precisely maps the location of the ovarian stem cell population, but it also suggests that this stem cell population might be the source of ovarian cancers. LGR5-expressing cells might be the source of malignancies of the reproductive tract, which makes these cells important from a clinical perspective. With respect to harvesting these cells and exploiting them for regenerative strategies, it is simply too early at this point to know if this is a realistic possibility, but perhaps this will be the next phase of Barker’s very interesting research.

Making Better Induced Pluripotent Stem Cells

On July 2nd of this year, a paper appeared in the journal Nature that performed complete genomic analyses of embryonic stem cells derived from embryos or cloned embryos, and induced pluripotent stem cells (iPSCs), which are made from reprogrammed adult cells.  They found that both embryonic stem cells made from cloned embryos and iPSCs derived from the same types of adult cells contained comparable numbers of newly introduced mutations.  However, when it came to the epigenetic modification of the genome (the small chemical tags attached to specific bases of DNA that gives the cell hints as to which genes to turn off), the epigenetic pattern of the embryonic stem cells made from cloned embryos more closely resembled that from embryonic stem cells.  The iPSCs still had some similarities with the adult cells from which they were derived whereas the embryonic stem cells made from cloned embryos were more completely reprogrammed.  From this the authors claimed that making embryonic stem cells by means of cloning is ideal for cell replacement therapies.

There is a big problem with this conclusion:  This was tried in animals and it did not work because of immunological rejection of the products from the stem cells.  For more information on this, see my book, The Stem Cell Epistles, chapter 18.

Despite this “bad news” for iPSCs, two recent papers have actually provided some good news for stem cells that can heal without destroying embryos.  The first paper comes from Timothy Nelson’s laboratory at the Mayo Clinic in Rochester, Minnesota.  Differentiation of iPSCs is, in some cases, rather efficient and the isolation procedures fail to effectively isolate the differentiated cells from potentially tumor-causing cells.  However, in other cases, the differentiation is inefficient and the isolation procedures are also rather poor, which leaves a large enough population of undifferentiated tumor-causing cells.

Nelson’ group has discovered that treating iPSCs and their derivatives with anti-cancer drugs like etoposide (a topoisomerase II inhibitor for those who are interested) increases engraftment efficiency and decreases the incidence of tumors.  My only problem with Nelson’s paper is that he and his colleagues used lentiviral vectors to make their iPSCs.  These vectors tend to produce iPSCs that are rather good at causing tumors.  I would have rather that he tried making iPSCs with other methods that do not leave permanent transgenes in the cells.  Nelson and his group transplanted their iPSC-derived cells into the hearts of mice where they could use high-resolution imaging to determine the number of cells that integrated into the heart and the presence of cell masses that were indicative of tumors.  None of the ectoposide-treated cell transplants caused tumors whereas 4 of the 5 transplants not treated with ectoposide caused tumors.  This paper appeared in Stem Cells and Development.

The second “good news” paper for iPSCs comes from Junji Takeda at the University of Osaka and Ken Igawa from the Tokyo Medical and Dental University, Japan.   In their paper from Stem Cells Translational Medicine, the Japanese groups collaborated to make iPSCs from skin based fibroblasts and then differentiate them into skin cells (keratinocytes).  However, they made the iPSCs in two different ways.  The first protocol utilized the piggyBac transposon system to make iPSCs.  The piggyBac system comes from moths, but it is highly active in mammalian cells.  It can deliver the genes to the cells, but the segment of DNA is then easily excised from the host cells without causing any mutations.  This system, therefore, will generate iPSCs that do not have any transgenes in them.  The second protocol used a system based on cytomegalovirus that leaves the transgenes in the cells but gradually inactivates their expression.

When these two types of iPSCs were compared, they seems to be essentially identical when grown in culture.  Thus in the pluripotent state, the cells were equivalent for the most part.  But once the iPSC lines were differentiated into skin cells, the transgene-free iPSCs formed skin cells that looked, behaved and had the same gene expression profile as normal human skin cells.  The transgene-containing iPSCs differentiated into skin cells, but they did not look quite like skin cells, did not have the same gene expression profile as normal human skin cells, and did not behave like normal human skin cells.

The moral of this story is that not all iPSC lines are created equally and the way you derive them is as important as the cell type from which they were derived.  Also, even incomplete differentiation does not need to be an obstacle for iPSCs, since the cancer-causing cells can be removed by means of specific drugs.  Finally, not all that glitters is gold.  Cloned embryos may give you stem cells that look more like embryonic stem cells, but so what.  These might still suffer from many of the same set backs.  Add to that the ethical problems with getting women to give up their eggs for research and cures (see Jennifer Lahl’s movie Eggsploitation for more disturbing information about that), and you have a losing combination.

StemCells, Inc., Sued by Former Employee Who Says Their Stem Cell Treatment is Unsafe

A California stem cell company, StemCells, Inc., that is developing cell-based therapies for several different neurological and eye conditions, is being sued by a former employee (whistleblower) who claims that the company did not follow proper protocols in the preparation of their treatments. Rob Williams, who was once a senior manager at StemCells, Inc., has alleged that the company fired him after he brought these problems to the attention of senior management.

According to the Courthouse New Service, Williams in the lawsuit stated that he “noted poor sterile technique, failure to adhere to current Good Manufacturing Practices in the company’s manufacturing process, and substantial deficiencies in the company’s Manual Aseptic Processing of HuCNS-SC (Human Central Nervous System Stem Cells) cell lines—failure and deficiencies that put patients at risk of infection or death during ongoing clinical trials.”

Ken Stratton, who serves as the general counsel for StemCells, Inc., has told the California Stem Cell Report that Williams’s employment “was terminated for performance deficiencies, and [the company] finds no merit to the allegations.” Stratton also said that “the elements of manufacturing practices that concerned Mr. Williams were immediately and carefully reviewed by the company.”

It might be worth noting that this lawsuit coincides with the departure this past April or May of StemCells, Inc.’s Executive VP of Manufacturing Operations and Regulatory Affairs, Stewart Craig, who took a position at Sangamo Biosciences.

Unfortunately for StemCells, Inc., this particular lawsuit comes soon after a second bit of bad press. Embryologist Alan Trounson led the California Institute for Regenerative Medicine (CIRM) until June of this year, but has joined the board of StemCells, Inc., shortly after leaving the state stem cell research funding agency. According to an opinion article written by Ron Leuty, who is a reporter for the San Francisco Business Times, Trounson has recused himself from discussions regarding a loan StemCells, Inc., received from CIRM in 2012 because of his close relationship with the company’s founder. “But the speed of his appointment to the StemCells board has raised questions” about a possible conflict of interest, Leuty wrote.

CIRM has been marred by conflicts of interest accusations since California voters in 2004 birthed CIRM through Proposition 71 and the subsequent sale of $3 billion in state bonds. Now it has one more strike against it.

Leuty called the situation an embarrassment for CIRM. “If the public perceives that individuals—researchers or CIRM employees or company executives—are feeding at the trough of the semiautonomous public agency, it isn’t going to help CIRM get more cash from that very same public that foots the bill.”

Patient-Specific Stem Cells Made More Easily?

A Michigan State University research team uncovered the function of an already characterized gene that could be linchpin in the derivation of patient-specific stem cells that might be able to save millions of lives by differentiating into practically any cell in the body.

The gene is known as ASF1A, and even though it was not discovered by the team, ASF1A is one of the genes responsible for the mechanism of cellular reprogramming. Cellular reprogramming de-differentiates adult cells into less mature stem cells that have the capacity to differentiate into any cell type in the adult body.

This work was published in the journal Science. In this paper, the MSU team analyzed more than 5,000 genes from a human egg (oocyte) and determines that ASF1A in combination with another gene known as OCT4 and another molecule were primarily responsible for reprogramming.

Human oocytes

“This has the potential to be a major breakthrough in the way we look at how stem cells are developed,” said Elena Gonzalez-Munoz, a former MSU post-doctoral researcher and first author of the paper. “Researchers are just now figuring out how adult somatic cells such as skin cells can be turned into embryonic stem cells. Hopefully this will be the way to understand more about how that mechanism works.”

An MSU team identified the thousands of genes expressed in oocytes in 2006. From this list of genes, the genes responsible for cellular reprogramming were then identified.

In 2007, a Japanese research team led by Shinya Yamanaka found that by introducing four other genes into adult cells, they could derive embryonic-like stem cells without the use of a human egg. These cells are called induced pluripotent stem cells, or iPSCs. “This is important because the iPSCs are derived directly from adult tissue and can be a perfect genetic match for a patient,” said Jose Cibelli, an MSU professor of animal science and a member of the team.

Apparently, ASF1A and OCT4 work in together in combination with a hormone-like substance that also is produced in the oocyte called GDF9 to facilitate the reprogramming process. “We believe that ASF1A and GDF9 are two players among many others that remain to be discovered which are part of the cellular-reprogramming process,” Cibelli said.

“We hope that in the near future, with what we have learned here, we will be able to test new hypotheses that will reveal more secrets the oocyte is hiding from us,” he said. “In turn, we will be able to develop new and safer cell-therapy strategies.”

Potential Marker Found for Stem Cell Population in the Inner Ear

Hearing loss is common as we get older. Presbycusis or age-related hearing loss results from the progressive death of sensory cells in the cochlea. Wouldn’t it be great if a stem cell population in the inner ear replaced these dying auditory sensory cells?

As it turns out, a stem cell population might exist in the mammalian inner ear and researchers from the UC Davis Comprehensive Cancer Center have identified a polysialylated glycoprotein that regulates neurodevelopment and is on the surface of cells in the adult inner ear. This glycoprotein acts as a marker of early cells in the inner ear and allows researchers to identify immature cells in the inner ear. This discovery was published in the journal Biochemical and Biophysical Research Communications and potentially opens the door to developing stem cell replacement treatments in the inner ear to treat certain hearing disorders.

“Hearing loss is a complex process and is usually regarded as irreversible,” said Frederic A. Troy II, principal investigator of the study from the UC Davis Comprehensive Cancer Center. “Finding this molecule in the inner ear that is known to be associated with early development may change that view.”

The existence of a marker for immature stem cells could make it possible to isolate neural stem cells from the adult inner ear in those people who suffer from hearing loss, culture and expand these cells in the laboratory, and then re-introduce them back into the inner ear as functioning neurons. These implanted cells might recolonize and establish themselves and improve hearing.

During development, certain glycoproteins (carbohydrate-protein linked molecules) are expressed on cell surfaces and serve critical functions essential to the normal growth and organization of the nervous system. One member of the class of cell-surface glycoproteins is an unusual molecule called polysialic acid or polySia. The large size of polySia allows it to fill spaces between cells and its strong electric charge repels other molecules. Therefore, polySia prevents cells from adhering or attaching to one another and thereby promotes cell movement to other areas.

Neural cell adhesion molecules (NCAMs) are also expressed on neuronal cell surfaces. As their name suggests, NCAMs help cells stick together and stay put. However, when NCAMs become modified with polysialic acid (or becomes “polysialylated”), the cells no longer adhere to other cells and are induced to migrate to new areas. Re-expression of the “anti-adhesive” polySia glycan on the surface of many adult human cancer cells facilitates their detachment, which enhances their metastatic spread.

Neural stem cells with these polySia-NCAMs on their cell surfaces play very important roles during embryonic development because these cells are able to travel throughout the body and differentiate into specialized cells. During adulthood, neural stem cells with polySia-NCAMs may migrate to injured areas and promote healing.

“The landscape of the cell surface of developing cells is decorated with a bewildering array of informational-rich sugar-protein molecules of which polysialylated NCAMs are of chief importance,” explained Troy. “During the life of a cell, these surface molecules are critical to cellular proliferation, self-renewal, differentiation and survival—essential processes for normal embryonic development and tissue regeneration in adults.”

It was already well-known that polySia-NCAM-expressing cells exist in the central nervous system, but Troy’s study is the first to document that they are also in the peripheral nervous system, and specifically in the spiral ganglia, those cluster of nerve cells in the inner ear that are essential to hearing.

Working with adult cells isolated from the inner ear spiral ganglia of guinea pigs, Troy and his team showed that these spiral ganglia cells expressed both polySia and NCAM. The polySia component was abundantly present on neural stem cells but was markedly reduced on mature cells. This implies that the polySia-NCAM complex is present on immature cells and can serve as a biomarker to identify these immature cells.

“Finding polySia-NCAM—a functional biomarker that modulates neuronal differentiation—on adult inner ear neural stem cells after differentiation gives researchers a ‘handle’ to identify and isolate these cells from among the many cells taken from a patient,” said Jan Nolta, director of the UC Davis Stem Cell Program and the university’s Institute for Regenerative Cures. “This discovery will enhance research into spiral ganglion neurons and may bring treatments closer to patients with hearing deficits.”

Lead author Park Kyoung Ho, a professor at Catholic University College of Medicine in Seoul, Korea, initiated the research for this article while on sabbatical leave in Troy’s laboratory at UC Davis. With his colleague and co-author, Yeo Sang Won, he is now planning clinical trials, based on the findings, in Korea with individuals who suffer from hearing disorders.

Stem Cells Aid Muscle Strengthing and Repair After Resistance Exercise

University of Illinois professor of Kinesiology and Community Health, Marni Boppart and her colleagues have published experiments that demonstrate that mesenchymal stem cells (MSCs) rejuvenate skeletal muscle after resistance exercise. These new findings, which were published in the journal Medicine and Science in Sports and Exercise, might be the impetus for new medical interventions to combat age-related declines in muscle structure and function.

Marni Boppart

Injecting MSCs into mouse leg muscles before several bouts of exercise that mimic resistance training in humans and result in mild muscle damage caused increases in the rate of muscle repair and enhanced the growth and strength of those muscles in exercising mice.

“We have an interest in understanding how muscle responds to exercise, and which cellular components contribute to the increase in repair and growth with exercise,” Boppart said. “But the primary goal of our lab really is to have some understanding of how we can rejuvenate the aged muscle to prevent the physical disability that occurs with age, and to increase quality of life in general as well.”

MSCs are found throughout the body, but several studies have established that MSCs from different tissue sources have distinct biological properties. Typically, MSCs can readily differentiate into bone, fat, and cartilage cells, but coaxing MSCs to form skeletal muscle has proven to be very difficult. MSCs usually form part of the stroma, which is the connective tissue that supports organs and other tissues.

Because of their inability to readily differentiate into skeletal muscle, MSCs probably potentiate muscle repair by “paracrine” mechanisms. Paracrine mechanisms refer to molecules secreted by cells that induce responses in nearby cells. Not surprisingly, MSCs excrete a wide variety of growth factors, cytokines, and other molecules that, according to this new study, stimulate the growth of muscle precursor cells, otherwise known as “satellite cells.” The growth of satellite cells expands muscle tissue and contributes to repair following muscle injury. Once activated, satellite cells fuse with damaged muscle fibers and form new fibers to reconstruct the muscle and enhance strength and restore muscle function.

“Satellite cells are a primary target for the rejuvenation of aged muscle, since activation becomes increasingly impaired and recovery from injury is delayed over the lifespan,” Boppart said. “MSC transplantation may provide a viable solution to reawaken the aged satellite cell.”

Unfortunately, satellite cells, even though they can be isolated from muscle biopsies and grown in culture, will probably not be used therapeutically to enhance repair or strength in young or aged muscle “because they cause an immune response and rejection within the tissue,” Boppart said. But MSCs are “immunoprivileged,” which simply means that they can be transplanted from one individual to another without sparking an immune response.

“Skeletal muscle is a very complex organ that is highly innervated and vascularized, and unfortunately all of these different tissues become dysfunctional with age,” Boppart said. “Therefore, development of an intervention that can heal multiple tissues is ideally required to reverse age-related declines in muscle mass and function. MSCs, because of their ability to repair a variety of different tissue types, are perfectly suited for this task.”

Mesenchymal Stem Cells Make Tendons on Fabricated Collagen

Ozan Akkus and his colleagues from the Department of Mechanical and Aerospace Engineering at Case Western Reserve University in Cleveland, Ohio has succeeded in making fibers made completely from the protein collagen. Why is this a big deal? Because it is so bloody hard to do.

In a paper published the journal Advanced Functional Materials, Akkus and others describe the generation of their three-dimensional collagen threads. This is the first time anyone has described the formation of such threads made purely from collagen.

Collagen is a very widely distributed protein in our bodies. It is the major structural component of tendons, and most connective tissues, and as a whole, collagen composes approximately one-third of all the protein in our bodies. There are almost 30 different types of collagen; some collagens for stiff fibers and others form flat networks that act as cushions upon which cells and other tissues can sit.

Collagen biosynthesis is very complicated and occurs in several steps. First, the collagen genes are transcribed into messenger RNAs that are translated by ribosomes into collagen protein. However, collagen proteins are made in a longer, inactive form that must undergo several types of modifications before it is usable.

Collagen synthesis begins in a compartment of the cell known as the endoplasmic reticulum, which is a series of folded membranes associated with the nuclear membrane. Within the endoplasmic reticulum, the end piece of the collagen protein, known as the signal peptide, is removed by enzymes called signal peptidases that clip such caps off proteins. Now particular amino acids within the collagen protein chains are chemically modified. The significance of these modifications will become clear later, but two amino acids, lysine and proline, and -OH or hydroxyl groups added to them. This process is called “hydroxylation,” and vitamin C is an important co-factor for this reaction. Some of the hydroxylysine residues have sugars attached to them, and three collagen protein chains now self-associate to form a “triple ɣ helical structure.” This “procollagen” as it is called, is shipped to another compartment in the cell known as the Golgi apparatus. Within the Golgi apparatus, the procollagen it is prepared to be secreted to the cell exterior. Once secreted, collagen modification continues. Other proteins of the collagen protein chains called “registration peptides” are clipped off by procollagen peptidase to form “tropocollagen.” Multiple tropocollagen molecules are then lashed together by means of the enzyme lysyl oxidase, which links hydroxylysine and lysine residues together in order to form the collagen fibrils. Multiple collagen fibrils form a proper collagen fiber. Variations on a theme are also available, since collagen can also, alternatively, attached to cell membranes by means of several types of proteins, including fibronectin and integrin.

Now, if the cells has to go through all that just to make a collagen fiber, how tough do you think it is to make collagen fiber in a culture dish? Answer – way hard. In order to make collagen threads, Akkus and his team had to use a novel method for mature collagen production, and then they compacted the collagen molecules by means of the mobility of these molecules in an electrical field. This “electrophoretic compaction” method also served to properly align the collagen molecules until they formed proper collagen threads. Biomechanical analyses of these fabricated collagen threads showed that they had the mechanical properties of a genuine tendon. Akkus’ group when one step further and showed that a device they designed with movable electrodes could fabricate continuous collagen threads (). Thus, Akkus and his crew showed that they could make as many collagen threads as they needed and that these threads worked like tendons (see here for video). Are these guys good or what?

A. Schematic of basic electro-chemical cell layout for collagen alignment; B. Polarized image confirming the alignment of ELAC; C. Human mesenchymal stem cells on ELAC threads at day 1 and day 14. Cell form a confluent layer on day 14. Scale bar: 0.5 mm.

Nest, Akkus and his gang seeded collagen threads with mesenchymal stem cells (MSCs) from bone marrow. Remarkably, these collagen thread-grown mesenchymal stem cells differentiated into tenocytes, which are the cells that made tendons. Normally, MSCs do not readily form tenocytes in the laboratory, and they do not easily make tendons. However, in this case, the MSCs not only differentiated into tenocytes and made tenocyte-specific proteins and genes, but they do so without the addition of exogenous growth factors; the collagen threads were all the cells needed.

The seeded MSCs made Collagen I, which is the most abundant collagen of the human body, and is present in scar tissue, tendons, skin, artery walls, corneas, the endomysium surrounding muscle fibers, fibrocartilage, and the organic part of bones and teeth. Other tendon-specific proteins that were made included tenomodulin, and COMP (Cartilage oligomeric matrix protein). Furthermore, the electrically-aligned collagen does a better job of inducing the tenocyte fate in MSCs than collagen that is randomly oriented.

These remarkable and fascinating results demonstrate scaffolds made of densely compacted collagen threads stimulates tendon formation by Mesenchymal stem cells. Thus electrically aligned collagen as a very promising candidate for functional repair of injured tendons and ligaments. Now it is time to show that this can work in a living creature. Let the preclinical trials commence!!

Stem Cell Treatment Saves Man’s Leg From Amputation

Clive Randell loves motorcycles, but an unfortunate accident in 2011 seriously injured his leg and potentially prevented him from ever riding his beloved Harley-Davidson motorcycle again. His leg had several open fractures and one particular fracture that left some bone that protruded through his skin. He had extensive skin loss, and his doctors told me several times that his leg would have to be amputated. Things looked grim to say the least.

However, new stem cell procedure that repairs severely fractured bones has healed his bad leg and saved Clive from amputation. In fact, now Clive can ride his motorbike again. This new, pioneering stem cell procedure could give a new hope for victims of severe accidents who face limb amputation.

This new procedure uses stem cells extracted from the patient’s bone marrow from the patient’s pelvis and then mixes these cells with a specially created gel matrix to provide the cells with the right environment in order for them to form bone. This stem cell/matrix was then injected into the damaged bone with some hardware, such as a rod, which is inserted into the bone for support. Over time, the stem cells regenerate bone at the fracture site, traversing the fracture with new bone and completely healing the damaged bone.

The intrepid physician who used this new procedure to heal Mr. Randall is Professor Anan Shetty, who serves as the Deputy Director of Minimally Invasive Surgery at Kent’s Canterbury Christ Church University. The motivation behind Dr. Shetty’s research is easy to understand. In the United Kingdom alone there are 350,000 serious fractures every year. Five to ten percent of these fractures are too extensive to heal and demand multiple surgeries, bone grafts, and other procedures that sometimes end in limb amputation if they fail to produce satisfactory results.

The fractured bone lacks an established blood supply, which means that it is very tough going for any implanted stem cells. Implanted stem cells have no way to receive signals to regenerate damaged cells. This new treatment circumvents this problem by using the bioengineered gel that contains the ingredients to that tells the stem cells what to do.

According to Professor Shetty, “Experiments have shown that collagen [gels] can trigger the transformation of stem cells into bone forming cells.” Dr. Shetty continued: “These “miracle” cells are abundant in bone marrow, so may be harvested, concentrated and applied with a collagen ‘scaffold’ into an area of poor healing.”

According to Clive Randall, “I may never dance the tango, but, thanks to Professor Shetty, I will be able to get as near to normal as possible.”

This bone-healing operation is performed under a general anaesthesia and only takes 30 minutes, after which the patient can walk out of the hospital and go home on the same day as the procedure. To date, six patients in the UK, four in India and 20 in South Korea have undergone this procedure.

Bone marrow for this procedure is drawn from the crest of the ilium of the patient’s pelvis by means of a stiff, hollow needle. Bone marrow contains a mixture of differ types of stem cells (including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells), and red blood cells. The bone marrow extract is then concentrated through centrifugation, but the red blood cells are usually removed. The concentrated bone marrow stem cell preparation is then mixed with collagen and this mixture is ready for implantation by injection.

For the surgical procedure, the surgeon stabilizes the fracture with a plate or long metal rod that is inserted through the central medullary canal or the bone. These stabilizing tools can be inserted with a small incision. The stem cell and collagen suspension is then injected into the fracture site and around the bone, guided by either live fluoroscopy or X-ray.

After the surgery, the patient is actually allowed put some weight on the affected limb, and is instructed to progressively increase the load he or she applies through his leg. Interestingly, Prof Shetty’s pioneering procedure cuts the healing time associated with these types of procedures in half, and at a cost of about $3,500 to $5,200, costs a fraction of the hundreds of thousands of dollars usually involved in amputation, rehabilitation and fitting the patient for prosthetics.

Professor Norimasa Nakamura, president elect of the International Cartilage Repair Society and one of the world’s leading authorities on stem cell treatment, has welcomed Prof Shetty’s work, saying: “It will revolutionize the whole field of bone fracture repairs. The patient has a more effective treatment and the health provider saves money. It’s a win-win situation.”

After his accident, Clive Randall, who worked as a high-altitude window cleaner who lived in Orpington, Kent, had a cage screwed to his damaged leg. He also underwent three bone grafts and several other procedures within 18 months after his accident. The accident also drastically changed his life. Even though the driver of the car was successfully prosecuted, he lost his job, girlfriend, and most of his money. He also had to take a deal of pain medication and became greatly depresses; at one time, understandably, he contemplated suicide. In a kind of “Hail Mary,” Clive turned to the internet and typed “I want to save my leg.”

He found Prof Shetty’s name, and the rest is history, but he is still in a state of disbelief over the reversal in his fortunes since having the operation in 2012. He said, “Six hours after the operation, Professor Shetty told me to get up and go for a walk. After being in and out of hospitals, I really couldn’t believe it. I’d suffered 15 months of being told there was a good chance I was going to lose my leg, yet eight weeks after the procedure I was told to start putting weight on it and to walk as much as I could. It still hurts to walk long distances, but that will improve. My foot is turned out a little bit to the side and I have a limp, but that’s a small price to pay to keep my leg. My hope is this procedure will eventually be available to everyone, since it can help so many people, particularly the military. The old way of mending broken bones is so painful and stops you getting on with your life. Professor Shetty’s stem cell surgery is quick and almost painless, so it’s important more people hear about it.”

There you have it from the patient himself.  Now only if the power-hungry, control-driven FDA would get off their duffs and look into bringing this procedure to the US?

Gene Therapy Creates a New Heart Pacemaker

When a patient’s heart beats too rapidly, too slowly or erratically, and if the usual heart medicines fail to properly regulate the heart rhythm, then the patient’s cardiologist may prescribe the implantation of an electronic pacemaker to regulate the heart rhythm. Even though implanted pacemakers are widely used, their installation requires an invasive surgery, they carry some risk of infection, and they also set off metal detectors during airport security checks. However, gene therapy might soon join the electronic pacemaker as a treatment for a poorly-regulated heart. It runs out that inserting a specific gene into heart-muscle cells can allow researchers to restore a normal heart rhythm in pigs, albeit temporarily.

Electronic pacemakers restore regular function to hearts by sending small electrical currents to the heart muscle in order to stimulate a heartbeat. This function is usually donned by the sinoatrial node, which is a cluster of a few thousand cardiac cells in the upper part of the right atrium that signals the heart to initiate a heartbeat and, therefore, sets the heart rate.

Heart Conducting System. 1) is the sinoatrial node or pacemaker and 2 is the atrioventricular node that receives the beat signal from the sinoatrial node and sends it to the ventricles.

A research team led by Eduardo Marbán, who is a cardiologist at Cedars-Sinai Medical Center in Los Angeles, California, attempted to engineer heart cells outside the sinoatrial node to act as the pacemaker of the heart. The findings from Marbán’s laboratory were reported in the journal Science Translational Medicine.

Marbán and his colleagues used 12 laboratory pigs for their laboratory experiments. In these animals, Marbán and others induced a fatal heart condition in which electrical activity that originates from the sinoatrial node cannot spread through the heart. This forces other, less capable parts of the heart to take over and act as a pacemaker. Then, Marbán’s group used high-frequency radiowaves to destroy the sinoatrial nodes in the pigs’ hearts. This caused the animals’ average heart rate to slow to about 50 beats per minute (compared to the normal rate of 100 or more beats per minute). Such animals, if they were a human, would require an electronic pacemaker.

Next, Marbán and other injected the pigs’ hearts with a genetically modified virus that carried a pig gene called Tbx18, which is involved in heart development. Within one day, infected heart cells infected with the virus began to express those genes usually found in sinoatrial node cells. These cells acted as the pacemaker and began to direct the pumping the heart at a normal rate. The animals maintained this steady beating for the two-week study period, whether resting, moving or sleeping.

In an interview, Marbán said that his method is simpler than other biological approaches to restore a normal heart rhythm to hearts. These other approaches include inducing cardiac muscle cells to a pluripotent state, then coaxing them to differentiate into pacemaker cells. However, Marbán cautioned that the effects of gene therapy might be temporary. Over time, the body’s immune system would probably recognize the virus used to deliver Tbx18 to the heart and attack and destroy the infected cells. Marbán’s team is presently monitoring pigs that have received the gene-therapy treatment for several months to measure the persistence of this pacemaker effect.

However, even if the treatment’s effects are limited, it could still prove useful, according to Marbán. For example, if a pacemaker patient suffers from an infection as a result of the pacemaker, that pacemaker must be temporarily removed. This patient could then receive a biological pacemaker that could keep the heart pumping steadily until the infection clears and a new device is implanted. The gene-therapy approach could also help unborn children with heart defects, or even children who quickly outgrow implanted pacemakers or people for whom surgery is simply too risky.

“I think it’s a truly creative idea,” says Ira Cohen, a cardiac electrophysiologist at Stony Brook University Medical Center in New York. He would like to see the therapy tested in dogs, whose average heart rate is 60-100 beats per minute, which is more similar to that of a human.

Marbán is presently in talks with the US Food and Drug Administration about developing a human trial, which he says could be just two to three years away.

Amniotic Fluid Stem Cells Aid Kidney Transplantation Success in a Pig Model

When a kidney patient receives a new kidney, the donated kidney undergoes a brief loss of blood supply followed by a restoration of the blood supply. This phenomenon is called ischemia/reperfusion (IR), and IR tends to cause cell death, followed by rather extensive scarring. Tissue scarring is called tissue fibrosis and a scarred kidney can lead to so-called transplant dysfunction, which means that the transplanted kidney does not work terrible well, and this can cause transplant failure.

Previous studies in laboratory rodents have shown that mesenchymal stem cells from amniotic fluid (afMSCs) are beneficial in protecting against transplant-induced fibrosis (Perin L, et al. PLoS One 2010;5:e9357; Hauser PV, et al. Am J Pathol 2010;177:2011-2021).

Now a research group at INSERM, France led by Thierry Hauet has developed a pig-based model of kidney autotransplantation that is comparable to the human situation with regards to the structure of the kidney and the damage that results from renal ischemia (for papers, see Jayle C, et al. Am J Physiol Renal Physiol 2007; 292: F1082-1093; and Rossard L, et al. Curr Mol Med 2012; 12: 502-505). On the strength of these previous experiments, Hauet’s group has published a new paper in Stem Cells Translational Medicine in which they report that porcine afMSCs can protect against IR-related kidney injuries in pigs.

Hauet and others showed that porcine afMSCs could be easily collected at birth and cultured. These cells showed the ability to differentiate into fat, and bone cells, made many of the same cell surface markers as other types of mesenchymal stem cells (e.g., CD90, CD73, CD44, and CD29), but showed a diminished ability to differentiate into blood vessel cells. When afMSCs are added to extirpated kidneys during the reperfusion (reoxygenation) process in an “in vitro” (fancy way of saying “in a culture dish”) model of organ-preservation, these stem cells significantly increased the survival of blood vessel (endothelial) cells. Endothelial cells are one of the main targets of ischemic injury, and the added cells bucked up these endothelial cells and rescued them from programmed cell death. In addition to these successes, Hauet and others showed that adding intact porcine afMSCs was not necessary, since addition of the culture medium used to grow the afMSCs (conditioned medium or CM) also rescued kidney endothelial cell death. The afMSC-treated kidneys survived because they had significantly larger numbers of blood vessels, and this seems to be the main factor that causes the extirpated kidney to survive intact.

While these experiments were successful, Hauet and others know that unless they were able to show that these cells improved kidney transplant outcomes in a living animal, their research would not be deemed clinically relevant. Therefore, Hauet and others injected afMSCs into the renal artery of pigs that had received a kidney transplant six days after the transplant. IR injuries following kidney transplants led to increased serum creatinine levels, but those pigs that had been infused with afMSCs showed reduced creatinine levels and lower protein levels in their urine (proteinuria). In fact, seven days after the stem cell infusion, the urine creatinine and protein levels had returned to pre-transplant levels. Three months after the transplant, the pigs were put down, and then the kidneys were subjected to tissue analyses. Microscopic examination of tissue slices from these kidneys showed that afMSC injection preserved the structural integrity of microscopic details of the kidneys and reduced the signs of inflammation. Control animals that were not treated with afMSCs showed disruption of the microscopic structures of the kidneys and extensive inflammation and scarring. Also, because the kidney controls blood chemistry, a comparison of the blood chemistries of these two groups of animals showed that the blood chemistries of the afMSC-treated animals were normal as opposed to the control animals.

Molecular analyses also showed a whole host of pro-blood vessel molecules in the kidneys of the afMSC-treated pigs. VEGFA (pro-angiogenic growth factor), and Ang1 (capillary structure strengthening and maintenance of vessel stability), proteins were increased in the kidneys of afMSC animals compared to control animals. Thus the infused stem cells increased the expression of pro-blood vessel molecules, which led to the formation of larger quantities of blood vessels, reduced cell death and decreased inflammation.

These findings demonstrate the beneficial effects of infused afMSCs on kidney transplant. Since afMSCs are easy to isolate and grow in culture, secrete proangiogenic and growth factors, and can differentiate into many cell lineages, including renal cells (see Perin L, et al. Cell Prolif 2007; 40: 936-948; De Coppi P, et al. Nat Biotechnol 2007; 25: 100-106; and In ‘t Anker PS, et al. Stem Cells 2004;22:1338-1345). This makes these cells a viable candidate for clinical application. This study also highlights pigs as a preclinical model as a powerful tool in the assessment of stem cell-based therapies in organ transplantation.

Patient-Specific Stem Cells Plus Personalized Gene Therapy for Blindness

Researchers from Columbia University Medical Center (CUMC) have devised protocols to develop personalized gene therapies for patients with an eye known as retinitis pigmentosa (RP), which is a leading cause of vision loss. While RP can begin during infancy, the first symptoms typically emerge during early adulthood. Typically the disease begins with night blindness, and RP eventually progresses to rob the patients of their peripheral vision. In its later stages, RP destroys photoreceptors in the macula, that region of the retina that provides the best vision under lighted conditions. RP is estimated to affect at least 75,000 people in the United States and 1.5 million worldwide.

The approach utilized by this Columbia team utilizes induced pluripotent stem (iPS) cell technology to transform patient’s skin cells into retinal cells, which are then used as a patient-specific model for disease study and preclinical testing.

The leader of this research group, Stephen H. Tsang, MD, PhD, showed that a form of RP caused by mutations to the MFRP gene compromised the structural integrity of the retinal cells. The MFRP gene encodes a protein called the Membrane Frizzled-Related Protein, which plays an important role in eye development. Mutations in the MFRP gene are associated with small eye conditions such as nanophthalmos, posterior microphthalmia, or retinal issues such as retinitis pigmentosa, foveoschisis, or even optic disc drusen. Tsang and his group, however, showed that the effects of these MFRP mutations could be reversed with gene therapy. Thus this new approach could potentially be used to create personalized therapies for other forms of RP, or even other genetic diseases.

“The use of patient-specific cell lines for testing the efficacy of gene therapy to precisely correct a patient’s genetic deficiency provides yet another tool for advancing the field of personalized medicine,” said Dr. Tsang, the Laszlo Z. Bito Associate Professor of Ophthalmology and associate professor of pathology and cell biology. This work was recently published in the online edition of Molecular Therapy, the official journal of the American Society for Gene & Cell Therapy.

Mutations in more than 60 different genes have been linked to RP. Such a genetic disease is known as a heterogeneous trait and genetic diseases like RP or deafness or other such conditions are very difficult to develop models to study. Animal models, though useful, have significant limitations because of interspecies differences. Eye researchers have also used human retinal cells from eye banks to study RP. This eye tissue comes from the eyes of patients who suffered from the disease and donated their eye tissue to research after death. Unfortunately, despite their usefulness, donated eye tissues typically illustrate the end stage of the disease process. Despite their usefulness, they reveal little about how RP develops. Also, there are no human tissue culture models of RP, since it is dangerous to harvest retinal cells from patients. Finally, human embryonic stem cells could be useful in RP research, but they are fraught with ethical, legal, and technical issues.

However, the Tsang group used iPS technology to transform skin cells from RP patients, each of whom harbored a different MFRP mutation, into retinal cells. Thus they created patient-specific models for studying the disease and testing potential therapies. Because they used iPS technology, Tsang found a way around the limitations and concerns and dog embryonic stem cells. Thus researchers can induce the patient’s own skin cells and de-differentiated them to a more basic, embryonic stem cell–like state. Such cells are “pluripotent,” which means that they can be transformed into specialized cells of various types.

When Tsang and others analyzed these patient-specific cells, they discovered that the primary effect of MFRP mutations is to disrupt the regulation of a cytoskeletal protein called actin, the scaffolding that gives the cell its structural integrity. “Normally, the cytoskeleton looks like a series of connected hexagons,” said Dr. Tsang. “If a cell loses this structure, it loses its ability to function.” They also found that MFRP works in tandem with another gene, CTRP5, and that a balance between the two genes is required for normal actin regulation.

In the next phase of the study, the CUMC team used adeno-associated viruses (AAVs) to introduce normal copies of MFRP into the iPS-derived retinal cells. This successfully restored the cells’ function. Tsang and others used gene therapy to “rescue” mice with RP due to MFRP mutations. According to Dr. Tsang, the mice showed long-term improvement in visual function and restoration of photoreceptor numbers.

“This study provides both in vitro and in vivo evidence that vision loss caused by MFRP mutations could potentially be treated through AAV gene therapy,” said coauthor Dieter Egli, PhD, an assistant professor of developmental cell biology (in pediatrics) at CUMC, who is also affiliated with the New York Stem Cell Foundation.

Dr. Tsang thinks this approach could potentially be used to study other forms of RP. “Through genome-sequencing studies, hundreds of novel genetic spelling mistakes have been associated with RP,” he said. “But until now, we’ve had very few ways to find out whether these actually cause the disease. In principle, iPS cells can help us determine whether these genes do, in fact, cause RP, understand their function, and, ultimately, develop personalized treatments.”

A Home A Stem Cell Could Love

In our bodies, stem cell populations live in specific places that are specially designed to accommodate them known as “stem cell niches.” Stem cell niches host and maintain stem cell populations, but the dependence of particular stem cells on their niche varies. For example, in the fruit fly, Drosophila melanogaster, the germ line stem cell niche can drive stem cells that have already begun to differentiate to revert into undifferentiated stem cells (see Brawley C and Matunis E. Science 2004;304:1331–4 and Kai T and Spradling A. Nature 2004;428:564–9). However, hair follicle stem cells do not revert when they return to their niche even if this niche has been depleted of stem cells (see Hsu Y-C, Pasolli HA, Fuchs E. Cell 2011;144:92–105). Also, blood cell-making stem cells that normally live in bone marrow can leave their niche in the bone marrow without losing their stem cell properties (Cao Y-A, et al., Proc Natl Acad Sci USA 2004;101:221–6). Finally, neural stem cells can exist and even self-renew outside their niche (Conti L, et al., PLoS Biol 2005;3:e283).

In order to properly grow stem cells in culture and manipulate them for therapeutic purposes, scientists have attempted to recapitulate stem cell niches in culture but only with very limited success.

Nevertheless, trying to get stem cells that have been introduced into a patient’s to engraft or make the new body their home has required a better understanding of stem cell niches.

To that end, Professor Claudia Waskow and her colleagues at the Technische Universität Dresden in Germany have utilized a downright ingenious method to make a mouse that can support the transplantation of human blood stem cells. This is despite the species barrier and, these mice do not need to have their own resident stem cell population obliterated with radiation.

How did Waskow and others do this? They used a mutation of a receptor called the “Kit receptor” to facilitate the engraftment of human cells. “What is the Kit receptor,” you ask? The Kit receptor is a protein in the membranes of blood stem cells that binds a soluble protein called stem cell factor (SCF). Stem cell factor drives certain types of blood cells to grow, and also mediates stem cells survival, proliferation and differentiation. Activation of the Kit receptor can also cause blood stem cells to leave the bone marrow and move into the peripheral circulation.

The Kit Receptor – AKA CD117

In the mouse model system designed by Waskow and others, the human blood stem cells grow and even differentiate into all blood-specific cell types without any additional treatment, and this includes the cells of the innate immune system. This is a milestone discovery because such cells normally do not form properly in “humanized” mice, but in Waskow’s experiment, these immune cells were efficiently generated. Significantly, these transplanted stem cells can be maintained in the mouse over a longer period of time compared to previously existing mouse models.

“Our goal was to develop an optimal model for the transplantation and study of human blood stem cells,” says Claudia Waskow, who recently took office of the professorship for “animal models in hematopoiesis” at the medical faculty of the TU Dresden. Before, coming to TU Dresden, Dr. Waskow was a group leader at the DFG-Center for Regenerative Therapies Dresden where most of the study was conducted.

Waskow’s team used a naturally occurring mutation of the Kit receptor and introduced it into her laboratory mice that lacked a functional immune system. This circumvented the two major obstacles of blood stem cell transplantation: the rejection by the recipient’s immune system and absence of free niche space for the incoming donor stem cells in the recipient’s bone marrow. Typically, the animal or the patient is treated with radiation to deplete the bone marrow of resident stem cells. This step, known as conditioning, creates usable space in the bone marrow for the implanted stem cells to take up residence and set up shop. However, irradiation is toxic a whole host of different cell types, not just bone marrow stem cells, and, unfortunately, has several strong side effects.

This Kit mutation in the mouse modifies the stem cell niche of the recipient mouse so that the resident stem cells are easily displaced by the human donor stem cells that possess a functional Kit receptor. This replacement works so well that irradiation was unnecessary, which allowed the study of human blood development in a physiological setting.

Waskow would like to use this new model system to study diseases of the human blood and immune system or to test new treatment options.

These data show that the Kit receptor (also known as CD117) is important for the function of human blood stem cells in a transplantation setting. Further work will concentrate on applying this new knowledge about the role of the receptor to improve conditioning therapy in bone marrow transplantation patients.

Gene Editing Does not Increase Mutation Rate in Stem Cells

Substituting one gene for another in cultured cells was once the stuff of science fiction, but with the ability to grow cells from our own bodies in culture and even convert them into embryonic-like stem cells, gene replacement has moved from the realms of science fiction to reality. However, the introduction of any all new technology comes with risks and trade-offs. In the case of gene replacement, there is the promise of fixing genes with mutations in them that cause genetic diseases. Unfortunately, any manipulation of the human genome runs the risk of adding new mutations to the genome whose side effects are unknown. Thus the cure might end up being worse than the disease itself.

New work from scientists at the Salk Institute in La Jolla, California has shown that new gene replacement techniques in stem cells does not increase the overall occurrence of mutations in those cultured cells. These new results were published the July 3 edition of the journal Cell Stem Cell.

“The ability to precisely modify the DNA of stem cells has greatly accelerated research on human diseases and cell therapy,” says senior author Juan Carlos Izpisua Belmonte, professor in Salk’s Gene Expression Laboratory. “To successfully translate this technology into the clinic, we first need to scrutinize the safety of these modified stem cells, such as their genome stability and mutational load.”

Introducing new genes into cells can occur by one of two methods. Engineered viruses can deliver new genes to a cell, which is then integrates the new DNA sequence in place of the old one. Alternatively, scientists can use custom targeted nucleases, such as TALEN proteins, which cut DNA at any desired location. Such proteins will extirpate the gene that needs to be replaced and then the new (potentially improved version of the gene) is simply added to the mix. The cell’s natural repair mechanisms will paste the new gene in place.

Belmonte’s lab has pioneered the use of modified viruses known as helper-dependent adenoviral vectors (HDAdVs) to fix genetic mutations that cause sickle-cell anemia. Sickle cell anemia is one of the most severe blood diseases found in the world. Belmonte and his collaborators have used HDAdVs to replace the mutant version of the globin gene in a stem cell line with a mutant-free version. This generated stem cells that could be theoretically be infused into patients’ bone marrow where they would create healthy blood cells.

Before such technologies are applied to humans, though, researchers must ascertain the risks of editing genes in stem cells. Even though both common gene-editing techniques have been shown to be accurate at altering the right stretch of DNA, concerns remain that the editing process could make the cells more unstable and prone to mutations in unrelated genes.

“As cells are being reprogrammed into stem cells, they tend to accumulate many mutations,” says Mo Li, a postdoctoral fellow in Belmonte’s lab and an author of the new paper. “So people naturally worry that any process you perform with these cells in vitro—including gene editing—might generate even more mutations.”

To test the safety of gene editing techniques, Belmonte’s research group, collaborated with BGI and the Institute of Biophysics, Chinese Academy of Sciences in China. They originally used a stem cell line that contains mutations in the beta-globin gene, which cause sickle-cell anemia. Belmonte then used HDAdV to edit the beta-globin genes of some cells, and edited the beta-globin genes of other cells by means of one of two TALEN proteins. Other cells were grown without any gene editing. Then, with the help of their Chinese collaborators, they fully sequenced the entire genome of each cell from the four edits and control experiment.

While all of the cells gained a low level of random gene mutations during the experiments, the cells that had undergone gene-editing—whether through HDAdV—or TALEN-based approaches—had no more mutations than the cells kept in culture.

“We were pleasantly surprised by the results,” Keiichiro Suzuki, a postdoctoral fellow in Belmonte’s lab and an author of the study, says. “People have found thousands of mutations introduced during iPSC reprogramming. We found less than a hundred single nucleotide variants in all cases.”

According to Li, this does not necessarily mean that there are no inherent risks to using stem cells with edited genes. However, it does mean that the editing process does not make stem cells that have undergone gene replacement are any less safe.

“We concluded that the risk of mutation isn’t inherently connected to gene editing,” he says. “These cells present the same risks as using any other cells manipulated for cell or gene therapy.” He adds that two other papers published in the same issue support their results (one by Johns Hopkins University and one from Harvard University and collaborators).

The next step for the Belmonte group is to determine if gene-repair in other cell types might be more likely to increase the mutation rate or if targeting other genes can cause unwanted mutations. They also hope that their findings will encourage those in the field to keep pursuing gene-editing techniques as a potential way to treat genetic diseases in the future.

Preserving Heart Tissue After a Heart Attack: Umbilical Cord-Coated Stem Cell Spheres

Eliana Martinez and her colleagues from the laboratories of Chuen Lee and Theo Kofidis at the National University of Singapore have published an extremely interesting paper in the journal Stem Cells and Development. In this paper, Martinez and her colleagues use a novel approach to deliver stem cells to the hearts of rats after a heart attack.

Usually, stem cells are given to heart attack patients in one of several ways. In laboratory animals, it is common to simply inject the stem cells directly into the heart muscle. This is done after the animals’ chest has been cut open. This procedure, known as a thoracotomy, is feasible in human patients, but unless the patient is undergoing coronary artery graft bypass surgery, cracking the chest leaves the patients in severe pain, greatly weakened, and with a very long recovery period. Therefore, unless necessary, this procedure is not preferred. Secondly, stem cells are delivered through the coronary arteries by means of the same technology used to deliver stents (percutaneous coronary intervention or PCI). In this case the cells are delivered through the coronary arteries while the arteries are propped open. This procedure is relatively easy to perform and no special equipment or training is required to deliver the cells, but several studies have shown that only a fraction of the cells make it to the heart muscle. The third technique uses direct injection into the heart muscle without cracking the patient’s chest. This technique uses special injection devices under the direction of sophisticated heart imaging technologies. Special equipment and specialized training is required to deliver the cells. Only a few centers offer this mode of delivery. The cells are well retained in the heart muscle, but a percentage of them leak out and find their way into the lung and other organs.

All of these techniques have their ups and downs. To that end, Martinez and her colleagues decided to deliver small spheres of stem cells surrounded by umbilical cord cells. These subamnion-cord-lining mesenchymal stem cell angiogenic spheroids (say that fast five times) consist of a special cell type from human umbilical cord called human umbilical cord vein endothelial cells or HUVECs that were used to encase another type of umbilical cord stem cell called cord-lining mesenchymal stem cells or CL-MSCs.

CL-MSCs have been evaluated in the laboratory and they seem to possess a robust ability to evade detection by the immune system and suppress inflammation, and do a better job of inducing healing than bone marrow-based stem cells (see Deuse T, et al., Cell Transplant. 2011;20(5):655-67). These cells also showed a marked ability to repair the heart after a heart attack (see Lilyana and others, Tissue Eng Part A 19:1303-1315).

To this end, Kofidis and his co-workers decided to use the spheroid technique because stem cells grown in liquid suspension and not flat culture dishes seem to do a better job of holding onto their healing properties than stem cell grown under standard conditions. Next, Martinez and others added HUVEC cells, which make blood vessels, the encase the CL-MSCs. Once they spheroids were made, they used fibrin (the protein found in blood clots) to paste the spheroids to the heart tissue after inducing a heart attack in laboratory rats.

These spheroids were mercifully called SASGs, since the proper name of these clusters was subamnion-cord mesenchymal stem cells angiogenic spheroids embedded within fibrin grafts (exhale). The laboratory animals were either given fibrin grafts without SASGs, neither fibrin grafts nor SASGs, and SASGs while the animal had its chest cracked, SASGs delivered without a thoracotomy (under video-assisted thoracoscopic surgery, and fibrin grafts under with no SASGs without have the chest cracked open.

In both cases in which SASGs were delivered, the structure and function of the heart improved in every physiological category examined. The heart beat more efficiently, the heart scar was smaller, there were more blood vessels, less, cell death, less sign of heart failure,

Even though this was a relatively small study in laboratory animals, it shows that a minimally invasive procedure can deliver stem cells to the heart that will stay in the heart and deliver healing to it,

This strategy should be expanded to larger numbers of animals and then, if it still statistically pans out, larger animal model systems should be examined (e.g., minipigs).   This is an ingenious technique, and hopefully, other laboratories will confirm the efficacy of this technique and the robust healing capabilities of this particular stem cell type from umbilical cord.

Heart Function Improved by Injecting Discarded Surgery Fat

Many patients with heart problems – such as heart disease or angina – may need to undergo cardiac surgery in order to restore or improve blood flow. But a new study suggests that the procedure may offer so much more; stem cells in fat discarded during cardiac surgery could be injected back into the patient’s heart to further improve its function.

A research team led by senior author Canadian cardiologist Dr. Ganghong Tian will present their findings at the Frontiers in Cardiovascular Biology meeting in Barcelona, Spain.

Previous work by this group has shown that subcutaneous fat (adipose tissue) contains stem cells that can reduce the severity of heart attacks, improve cardiac function, and augment blood vessel regeneration in laboratory animals with experimentally induced heart attacks. These fat-based stem cells can be easily obtained through liposuction. However, Tian noted, “But obtaining these from a patient undergoing cardiac surgery requires pre-surgery to collect adipose tissue from the subcutaneous region.”

Is there a better way? According to Tian, during cardiac surgery, the surgeon often removes fat tissue that resides around the heart (so-called mediastinal fat) in order to properly expose the heart. Tian wondered if this fat contain stem cells that could be re-introduced to the heart to improve its function after heart surgery

In order to test this hypothesis, Tian and others collected mediastinal fat tissue from 24 patients who had undergone cardiac surgery. Then Tian’s group injected rats with mediastinal fat stem cells. The rats injected with stem cells from mediastinal fat showed greater ventricular movement in their hearts and no reduction in left ventricular ejection fraction.

Closer examination of the stem cells from mediastinal fat showed that mediastinal fat housed a rather robust number of stem cells, and that these stem cells could differentiate into fat and bone cells. Also, these stem cells expressed genes that are often found in heart muscle cells.

With this pre-clinical information in hand, Tian and others examined the use of mediastinal fat-based stem cells in 13 rats with congestive heart failure. These stem cells were directly injected into the hearts of eight rats, and five were injected with a saline solution.

After 6 weeks, all the rats underwent magnetic resonance imaging (MRI). When the five control rats were compared with those who those rats that received injections of mediastinal fat-based stem cells, the stem cell-injected rats demonstrated greater ventricular movement in their hearts and no reduction in left ventricular ejection fraction (ejection fraction measures how much blood is being pumped out of the left ventricle of the heart).

Commenting on the team’s findings, Dr. Tian says: “This is the first evidence that stem cells collected from the mediastinal fat region are cardioprotective. They displayed the same cardioprotective capacity we found in our previous research on stem cells from subcutaneous fat tissue. This raises the exciting possibility of using a patient’s own stem cells, isolated from waste tissue during cardiac surgery, to improve their heart function.”

Tian noted that there are currently some issues with this procedure that need to be addressed with further research. Techniques must be developed to quickly isolate stem cells from mediastinal fat so they can be injected back into a patient’s heart during cardiac surgery. Tian said, “It currently takes several hours to purify the cells and we are looking for collaborators to help us devise a more efficient method.”

Tina and others would also like to examine the ability of these stem cells to improve cardiac function long-term, beyond the 6 weeks monitored in this study. Furthermore, Tian and his group would like to induce the stem cells into functional heart muscle cells that display electrical pulses and beating.

STAP Papers Retracted

The two papers that appeared in the journal Nature that described the derivation of embryonic stem cell-like cells simply by exposing cells to environmental stresses have been formally retracted. In a notice of retraction from the Riken Center’s Haruko Obokata, who was the lead author of these papers, and her colleagues said that “[s]everal critical errors have been found in our Article and Letter.” The notice also pointed out that a subsequent investigation of those errors by an internal Riken Center investigation found evidence of research misconduct.

“The STAP technology, indeed, sounded too good to be true,” said Dusko Ilic, from King’s College London, to the Reuters news group. “I hoped that Haruko Obokata would prove at the end all those naysayers wrong. Unfortunately, she did not.”

In an editorial that appeared in Nature, Ivan Oransky from a blog site known as Retraction Watch, argue that it couldn’t have caught the errors. Oransky wrote: We at Nature have examined the reports about the two papers from our referees and our own editorial records,” the editorial notes. “Before publishing, we had checked that the results had been independently replicated in the laboratories of the co-authors.” Nevertheless, the journal says this incident has highlighted flaws in the peer-review publishing process.

“We — research funders, research practitioners, institutions and journals — need to put quality assurance and laboratory professionalism ever higher on our agendas, to ensure that the money entrusted by governments is not squandered, and that citizens’ trust in science is not betrayed,” it adds.

The simple fact is that reviewers examine data, figures and materials and methods, but they have no gift of ESP to determine is the authors are telling the truth.  Truth-telling and honesty are virtues without which science cannot exist.  What is the basis of honesty and truth-telling?  Well, the secular, pragmatic worldview would suggest that truth-telling works and without it we cannot do science without it.  However, if truth-telling gets the individual scientist ahead for a time, then why shouldn’t they prevaricate?  What should the individual worry about what the collective thinks or needs?

It is at this point that I must interject that the Christian worldview provides the foundation for honesty and truth-telling.  The Christian tells the truth because God is the author of all truth and is by His very nature, the truth (see John 14:6).  To not tell the truth is to dishonor God and not live in accordance with his revealed prescriptions.  Therefore, the Christian worldview explains why we should tell the truth when reporting our experiments.