Fat-Based Stem Cell Product HemaXellerate Will be Tested in Clinical Trials for Aplastic Anemia


A regenerative medicine company called Regen BioPharma, Inc., has announced that it received a communication from the U.S. Food and Drug Administration that grants it permission to initiate clinical trials under its Investigational New Drug (IND) #15376.

Granting of the IND gives the green light to Regen BioPharma to begin testing their product HemaXellerate in clinical trials with human patients. HemaXellerate is a personalized stem cell treatment for patients whose bone marrow no longer works (aplastic anemia). It uses fat-based stem cells from a patient’s own belly fat to treat bone marrow that has been damaged. HemaXellerate uses the patient’s own fat-based stem cells as a source of endothelial (blood vessel) cells to heal damaged bone marrow.

Aplastic anemia occurs when the bone marrow stops producing sufficient numbers of blood cells. It is a potentially fatal disease of the bone marrow that leads to bleeding, infection and fever. Patients with severe or even very severe aplastic anemia have a mortality rate of greater than 70%. Current treatments for aplastic anemia include blood transfusions, immunosuppression and stem cell transplantation.

This Phase I clinical trial will treat patients who have been diagnosed with refractory aplastic anemia, which includes those patients with aplastic anemia who were unsuccessfully treated with first-line immunosuppressive therapy. Patients treated with HemaXellerate with be followed for safety parameters and signals of treatment efficacy. Since this will be an unblinded trial, all data will be available as the study progresses.

“Current drug-based approaches for healing bone marrow dysfunction involve flooding the body with growth factors, which is extremely expensive and causes unintended consequences because of lack of selectivity,” said Harry Lander, Ph.D., President and Chief Scientific Officer of Regen Biopharma. “By utilizing a cell-based approach that both modulates the immune system and stimulates production of blood cells, we aim to offer alternatives to the current approaches to treating patients with aplastic anemia. This product will complement our immune-modulatory pipeline that includes a potential novel checkpoint inhibitor.”

If HemaXellerate passes this clinical trial, Regen Biopharma would like to position HemaXellerate as a treatment for bone marrow dysfunction on par with other members of the hematopoietic growth factor market that includes drugs such as Neupogen®, Neulasta®, Leukine® and Revolade®.

“The FDA clearance marks a substantial step for Regen, in that we are now a clinical-stage company. We are grateful to our collaborators and scientific advisory board members who have worked tirelessly in bringing our product to the point where the FDA has permitted treatment of patients,” said David Koos, Ph.D., Chairman and Chief Executive Officer of Regen BioPharma. “We believe the success of today will not only allow for the rapid execution of HemaXellerate’s development plan, but will also allow for more rapid translation of the company’s other immune modulatory products to the clinic.”

International Stem Cell Corp’s Parthenogenetic Stem Cells to Be Used in A Clinical Trial to Treat Parkinson’s Disease Patients


The Australian government has recently given its approval for a clinical trial of what is almost certainly a medical first. The Carlsbad-based stem cell company, International Stem Cell Corp. (ISCO), a publicly traded biotechnology company, has developed a unique stem cell technology to address particular conditions.

The clinical trial that has been approved will examine the use the ISCO’s unique stem cell products in the treatment of Parkinson’s disease. Twelve Parkinson’s patients will receive implantations of these cells sometime in the first quarter of 2016, according to Russell Kern, ISCO’s chief scientific officer. The implanted cells will be neural precursor cells, which are slightly immature neurons that will complete their maturation in the brain, hopefully into dopamingergic neurons, which are the precise kind of neurons that die off in patients with Parkinson’s disease.

Parkinson’s disease (PD) is a progressive disorder of the nervous system that affects voluntary movement. PD develops gradually and sometimes begins with a slight tremor in only one hand, but PD may also cause stiffness or slowing of movement. PD worsens over time.

PD patients suffer from tremor, or shaking of the limbs, particularly when it is relaxed and at rest. Over time, PD reduces the ability to move and slows movement (bradykinesis) which makes simple tasks difficult and time-consuming. Muscle stiffness may occur and this limits the range of motion and causes pain. PD patients also suffer from stooping posture and balance problems and a decreased ability to perform unconscious movements. For example, they have trouble swinging their arms while they walk, blinking, or smiling. They might also experience speech problems that can range from slurring of the speech to monotone speech devoid of inflexions, or softer speech with hesitations before speaking. Writing might also become problematic.

PD is caused by the gradual death of neurons in the midbrain that produce a chemical messenger called dopamine. The drop in dopamine levels in the system of the brain that controls voluntary movement leading to the signs and symptoms of Parkinson’s disease.

Several different animal experiments with a variety different cell types have established that transplantation to dopamine-making neuronal precursors into the midbrains of laboratory animals with artificially-induced PD can reverse the symptoms of PD. Dopaminergic neurons can be derived from embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), umbilical cord blood hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), and NSCs (see Petit G. H., Olsson T. T., Brundin P. Neuropathology and Applied Neurobiology. 2014;40(1):60–67). Also, since the 1980s, various cell sources have been tested, including autografts of adrenal medulla, sympathetic ganglion, carotid body-derived cells, xenografts of fetal porcine ventral mesencephalon, and allografts of human fetal ventral mesencephalon (fVM) tissues have been implanted into the midbrains of PD patients (Buttery PC, Barker RA. J Comp Neurol. 2014 Aug 15;522(12):2802-16). While the results of these trials were varied and not terribly reproducible, these studies did show that the signs and symptoms of PD could be reversed, in some people, by implanting dopamine-making neurons into the midbrains of PD patients.

ISCO has derived neural precursor cells from a completely new source. ISCO scientists have taken unfertilized eggs from human egg donors and artificially activated them so that they self-fertilize, and then begin dividing until they form a blastocyst-stage embryo from which stem cells are derived. This new class of stem cells, which were pioneered by ISCO, human parthenogenetic stem cells (hpSCs) have the best characteristics of each of the other classes of stem cells. Since these stem cells are created by chemically stimulating the oocytes (eggs) to begin division, the oocytes are not fertilized and no viable embryo is created or destroyed. This process is called parthenogenesis and parthenogenetic stem cells derived from the parthenogenetically-activated oocytes, are produced from unfertilized human egg cells.

The stem cells are created by chemically stimulating the oocytes (eggs) to begin division.  The oocytes are not fertilized and no viable embryo is created or destroyed.
The stem cells are created by chemically stimulating the oocytes (eggs) to begin division. The oocytes are not fertilized and no viable embryo is created or destroyed.

Why did ISCO decide to do this trial in Australia? According to Kern, ISCO chose to conduct their clinical trial in Australia because its clinical trial system is more “interactive,” which allows for better collaboration with Australia’s Therapeutic Goods Administration on trial design. This clinical trial, in fact, is the first stem cell trial for PD according to the clinical trial tracking site clinicaltrials.gov. The test will be conducted by ISCO’s Australian subsidiary, Cyto Therapeutics.

The approach pioneered in this clinical trial might cure or even provide an extended period of relief from the symptoms of PD. If this clinical trial succeeds, the stem cell clinical trial dam might very well break and we will see proposed clinical trials that test stem cell-based treatments for other neurodegenerative diseases such as Huntington’s disease, Lou Gehrig’s disease (ALS), frontotemporal dementia, or even Alzheimer’s disease.

ISCO has spent many years developing their parthenogenetic technology with meager financing. However the company’s total market value amounts to something close to $11.1 million, presently.

hpSCs are pluripotent like embryonic stem cells. Because they are being used in the brain, they will not be exposed to the immune system. Therefore an exact tissue type match is not necessary for this type of transplantation. In their publications, ISCO scientists have found their cells to be quite stable, but other research groups who have worked with stem cells derived from parthenogenetically-activated embryos have found such cells to be less stable than other types of pluripotent stem cells. The stability of the ISCO hpSCs remains an open question. The lack of a paternal genome might pose a safety challenge for the use of hpSCs.

Rita Vassena and her colleagues in the laboratory of Juan Carlos Izpisua Belmonte at the Salk Institute for Biological Studies in La Jolla, CA examined the gene expression patterns of mesenchymal stem cells derived from hpSCs and found that the overall gene expression patterns were similar to MSCs made from embryonic stem cells or induced pluripotent stem cells. However, upon further differentiation and manipulation, the gene expression patterns of the cells began to show more variability and further depart from normal gene expression patterns (Vassena R, et al Human Molecular Genetics 2012; 21(15): 3366-3373). Therefore, the derivatives of hpSCs might not be as stable as cellular derivatives from other types of stem cells. The good news about hpSCs established from parthenogenetic ESCs were reported to be morphologically indistinguishable from embryonic stem cells derived from fertilized embryos, and seem to show normal gene expression or even correct genomic imprinting in chimeras, when pESCs were used in tissue contribution (T.Horii, et al Stem Cells, vol. 26, no. 1, pp. 79–88, 2008).

For those of us who view the early embryo as the youngest members of the human community who have the right not to be harmed, hpSCs made by ISCO remove this objection, since their derivation does not involve the death of any embryos.

The ISCO approach to Parkinson’s is similar to that of a San Diego group called Summit for Stem Cell, which is going to use induced pluripotent stem cell derivatives. This nonprofit organization is presently raising money for a clinical trial to test the efficacy of their treatment.

Both groups intend to transplant the cells while they are still slightly immature, so that they can complete their development in the brain. Animal studies suggest that implanting immature precursors are better than transplanting mature dopaminergic neurons into the midbrain. The precursors then differentiate into dopamine-making neurons, and other cells differentiate into supportive glial cells, which support the dopamine-making neurons.

“It’s a dual action,” Kern said. “Also, neural stem cells reduce inflammation, and inflammation is huge in Parkinson’s.”

Summit 4 Stem Cell will also take a similar approach, according to stem cell scientist Jeanne Loring, a leader of the Summit 4 Stem Cell project. The cells make proper connections with the brain better when they are still maturing, said Loring, who’s also head of the regenerative medicine program at The Scripps Research Institute in La Jolla. This is all provided that Summit 4 Stem Cell can raise the millions of dollars required for the clinical trial and secure the required approvals from the U.S. Food and Drug Administration.

Loring said she views ISCO as a partner in fighting Parkinson’s. One of her former students is working for the company, she said. “The whole idea is to treat patients by whatever means possible,” Loring said.

ISCO’s choice of Australia for its streamlined regulatory process makes sense, Loring said. Her team, with U.S.-based academics and medical professionals, doesn’t have the same flexibility as ISCO in looking for clinical trial locations, she said.

A Stem Cell Treatment for Hair Loss


Male and female pattern baldness involves the receding of the hair-line, hair loss and the thinning the hair covering on the scalp. Hair loss is also called alopecia and is a common problem among the elderly, those afflicted with certain diseases of the scalp, or those who take certain medicines. Hair life is not the end of life, but it can change someone’s appearance, affect their self-image, and affect someone’s emotional state. People with hair loss can use topical monoxidil (Rogaine), take an oral drug called finasteride (Propecia), or undergo a hair transplant. The drugs, however, must be used constantly or they stop working and hair transplants are horribly expensive.

Can stem cell-based treatment restore hair after it has been lost? Fortunately, a stem cell population called dermal papilla cells (DPCs), which are a type of mesenchymal stem cell population in hair follicles, have been identified and even characterized to some extent. DPCs are responsible for the formation of hair-follicles and play a very role in the process of hair cycling in which the hair shaft grows, is shed, is reestablished, and grows again. DPC might be useful for treating alopecia, but they do not survive when cultured outside the body, and this limitation has limited developing stem cell-based treatments for hair loss.

Now collaboration between two scientific research teams from Canada and China have resulted in a new way to use stem cells to treat hair loss.

A research team from the Nanfang Hospital of Southern Medical University, China, led by Zhi-Qi Hu and a Canadian team from the University of Manitoba, Canada, led by Malcolm Xing have designed a three-layered tunic that feeds and protects the DPCs and allows them to grow outside the body.

Xing describes this nutritive tunic as a “nutritious nano-clothing,” made of gelatin and alginate. These molecules can self-assemble and Hu and Xing and their coworkers encapsulated the cells within an inner layer of gelatin, a middle layer of alginate loaded with fibroblast growth factor-2, and an outer layer of gelatin. They call this method of encapsulation “layer-by-layer (LBL) nano-coating.”  This gelatin/alginate coating creates a protective microenvironment for cultured DPC and provides them with a significant source of a growth factor called fibroblast growth factor-2 (FGF2), which enhances proliferation of the DPCs and induces hair cell fates. The use of these three-layered tunics keeps the inductive signals close to the DPCs and circumvents the difficulties encountered in regenerating new hair follicles on bald skin.

DPC - hair follicle regeneration

When the Xing and Hu teams implanted these encased DPCs into the skin of nude mice, the implanted encapsulated cells generated the growth of abundant hair. The hair produced by these cells also was rooted in hair follicles that were normal in their appearance and function. The coating improvised by these teams greatly augmented the therapeutic capabilities of the DPCs by recapitulating the niche in which these cells are normally found. This stem cell niche induces the cells to secrete the native extracellular matrix that typically surrounds the cells and release the growth factors that keep the cells growing and in the proper stage of the cell cycle.

According to Xing, the most difficult part of this research project was “optimizing the concentrations of the coated polymers and manufacturing conditions to make the cells happy and healthy.”

Regenerative medicine researcher Oommen Varghese, from Uppsala University in Sweden, who was not involved in this work, said, “This is fascinating science that has enormous potential for clinical translational of stem cell based regenerative medicine. Such a coating could also protect cells from innate immunity, thereby improving the in vivo survivability. This is a major challenge in stem cell based translational research.”

Xing and his collaborators and colleagues would like to transform this technique from a laboratory bench to a clinical application that can be tested in human clinical trials.

ASTIC Clinical Trial Fails to Show Clear Advantage to Hematopoietic Stem Cell Transplantation as a Treatment for Crohn’s Disease


Patients with Crohn’s disease (CD) sometimes suffer from daily bouts of stomach pain and diarrhea. These constant gastrointestinal episodes can prevent them from absorbing enough nutrition to meet their needs, and, consequently, they can suffer from weakness, fatigue, and a general failure to flourish.

To treat Crohn’s disease, physicians use several different types of drugs. First there are the anti-inflammatory drugs, which include oral 5-aminosalicylates such as sulfasalazine (Azulfidine), which contains sulfur, and mesalamine (Asacol, Delzicol, Pentasa, Lialda, Apriso). These drugs, have several side effects, but on the whole are rather well tolerated. If these don’t work, then corticosteroids such as prednisone are used. These have a large number of side effects, including a puffy face, excessive facial hair, night sweats, insomnia and hyperactivity. More-serious side effects include high blood pressure, diabetes, osteoporosis, bone fractures, cataracts, glaucoma and increased chance of infection.

If these don’t work, then the stronger immune system suppressors are brought out. These drugs have some very serious side effects. Azathioprine (Imuran) and mercaptopurine (Purinethol) are two of the most widely used of this group. If used long-term, these drugs can make the patient more susceptible to certain infections and cancers including lymphoma and skin cancer. They may also cause nausea and vomiting. Infliximab (Remicade), adalimumab (Humira) and certolizumab pegol (Cimzia) are the next line of immune system suppressors. These drugs are TNF inhibitors that neutralize an immune system protein known as tumor necrosis factor (TNF). These drugs are also associated with certain cancers, including lymphoma and skin cancers. The next line of drugs include Methotrexate (Rheumatrex), which is usually used to treat cancer, psoriasis and rheumatoid arthritis, but methotrexate also quells the symptoms of Crohn’s disease in patients who don’t respond well to other medications. Short-term side effects include nausea, fatigue and diarrhea, and rarely, it can cause potentially life-threatening pneumonia. Long-term use can lead to bone marrow suppression, scarring of the liver and sometimes to cancer. You will need to be followed closely for side effects.

Then there are specialty medicines for patients who do not respond to other medicines or who suffer from openings in their lower large intestines to the outside world (fistulae). These include cyclosporine (Gengraf, Neoral, Sandimmune) and tacrolimus (Astagraf XL, Hecoria). These have the potential for serious side effects, such as kidney and liver damage, seizures, and fatal infections. These medications are definitely cannot be used for long period of time as their side effects are too dangerous.

If the patient still does not experience any relief, then two humanized mouse monoclonal antibodies natalizumab (Tysabri) and vedolizumab (Entyvio). Both of these drugs bind to and inhibit particular cell adhesion molecules called integrins, and in doing so prevent particular immune cells from binding to the cells in the intestinal lining. Natalizumab is associated with a rare but serious risk of a brain disease that usually leads to death or severe disability called progressive multifocal leukoencephalopathy. In fact, so serious are the side effects of this medicine that patients who take this drug must be enrolled in a special restricted distribution program. The other drug, vedolizumab, works in the same way as natalizumab but does not seem to cause this brain disease. Finally, a drug called Ustekinumab (Stelara) is usually used to treat psoriasis. Studies have shown it’s useful in treating Crohn’s disease and might useful when other medical treatments fail. Ustekinumab can increase the risk of contracting tuberculosis and an increased risk of certain types of cancer. Also there is a risk of posterior reversible encephalopathy syndrome. More common side effects include upper respiratory infection, headache, and tiredness.

If this litany of side effects sounds undesirable, then maybe a cell-based treatment can help Crohn’s patients. To that end, a clinical trial called the Autologous Stem Cell Transplantation International Crohn’s Disease or ASTIC trial was conducted and its results were published in the December 15th, 2015 edition of the Journal of the American Medical Association.

The ASTIC trial enrolled 45 Crohn’s disease patients, all of whom underwent stem cell mobilization with cyclophosphamide and filgrastim, and were then randomly assigned to immediate stem cell transplantation (at 1 month) or delayed transplantation (at 13 months; control group).  Blood samples were drawn and mobilized stem cells were isolated from the blood.  In twenty-three of these patients, their bone marrow was partially wiped out and reconstituted by means of transplantations with their own bone marrow stem cells. The other 22 patients were given standard Crohn disease treatment (corticosteroids and so on) as needed.

The bad news is that hematopoietic stem cell transplantations (HSCT) were not significantly better than conventional therapy at inducing sustained disease remission, if we define remission as the patient not needing any medical therapies (i.e. drugs) for at least 3 months and no clear evidence of active disease on endoscopy and GI imaging at one year after the start of the trial. All patients in this study had moderately to severely active Crohn’s disease that was resistant to treatment, had failed at least 3 immunosuppressive drugs, and whose disease that was not amenable to surgery.  All participants in this study had impaired function and quality of life.  Also, the stem cell transplantation procedure, because it involved partially wiping out the bone marrow, cause considerable toxicities.

Two patients who underwent HSCT (8.7%) experienced sustained disease remission compared to one control patient (4.5%). Fourteen patients undergoing HSCT (61%) compared to five control patients (23%) had discontinued immunosuppressive or biologic agents or corticosteroids for at least 3 months. Eight patients (34.8%) who had HSCTs compared to two (9.1%) patients treated with standard care regimens were free of the signs of active disease on endoscopy and radiology at final assessment.

However, there were 76 serious adverse events in patients undergoing HSCT compared to 38 in controls, and one patient undergoing HSCT died.

So increased toxicities and not really a clear benefit to it; those are the downsides of the ASCTIC study.  An earlier report of the ASTIC trial in 2013, while data was still being collected and analyzed was much more sanguine.  Christopher Hawkey, MD, from the University of Nottingham in the United Kingdom said this: “Some of the case reports are so dramatic that it’s reasonable to talk about this being a cure in those patients.”  These words came from a presentation given by Dr. Hawkey at Digestive Disease Week 2013.  Further analysis, however, apparently, failed to show a clear benefit to HSCT for the patients in this study.  It is entirely possible that some patients in this study did experience significant healing, but statistically, there was no clear difference between HSCT and conventional treatment for the patients in this study.

The silver lining in this study, however, is that compared to the control group, significantly more HSCT patients were able to stop taking all their immunosuppressive therapies for the three months prior to the primary endpoint. That is a potential upside to this study, but it is unlikely for most patients that this upside is worth the heightened risk of severe side effects. An additional potential upside to this trial is that patients who underwent HSCT showed greater absolute reduction of clinical and endoscopic disease activity. Again, it is doubtful if these potential benefits are worth the higher risks for most patients although it might be worth it for some patients.

Therefore, when HSCT was compared with conventional therapy, there was no statistically significant improvement in sustained disease remission at 1 year. Furthermore, HSCT was associated with significant toxicity. Overall, despite some potential upside to HSCT observed in this study, the authors, I think rightly, conclude that their data do not support the widespread use of HSCT for patients with refractory Crohn’s disease.

Could HSCT help some Crohn’s patients more than others? That is a very good question that will need far more work with defined patient populations to answer.  Perhaps further work will ferret out the benefits HSCT has for some Crohn’s disease patients relative to others.

The ASTIC trial was a collaborative project between the European Society for Blood and Marrow Transplantation (EBMT) and the European Crohn’s and Colitis Organization (ECCO) and was funded by the Broad Medical Foundation and the Nottingham Digestive Diseases Centers.

Adult Directly Reprogrammed With Proteins into Cardiac Progenitor Cells Heal Heart After a Heart Attack and Make New Heart Muscle


Jianjun Wang from Wayne State School of Medicine in Detroit, Michigan and Xi-Yong Yu from Guangzhou Medical University and a host of graduate students and postdoctoral research fellows in their two laboratories have teamed up to make human cardiac progenitor cells (CPCs) from human skin fibroblasts through direct reprogramming. Direct reprogramming does not go through a pluripotent intermediate, and, therefore, produces cells that have a low chance of generating tumors.

To begin their study, Wang, and Yu and their colleagues isolated fibroblasts from the lower regions of the skin (dermis) and grew them in culture. Then they reprogrammed these cells in a relatively novel manner. This is a little complicated, but I will try to keep it simple.

Reprogramming cells usually requires scientists to infect cells with recombinant viruses that have been genetically engineered to express particular genes in cells or force cells to take up large foreign DNA. Both of these techniques can work relatively well in the laboratory, but you are left with cells that are filled with foreign DNA or recombinant viruses. It turns out that directly reprogramming cells only requires transient expression of specific genes, and once the cells have recommitted to a different cell fate, the expression of the genes used to get them there can be diminished.

To that end, some enterprising scientists have discovered that inducing cells to up modified proteins can also reprogram cells. Recently a new reagent called the QQ-reagent system can escort proteins across the cell membrane. The QQ-reagent has been patented and can sweep proteins into mammalian cells with high-efficiency and low toxicity (see Li Q, et al (2008) Methods Cell Biol 90:287–325).

Wang and Yu and their coworkers used genetically engineered bacteria to overexpress large quantities of four different proteins: Gata4, Hand2, Mef2c, and Tbx5. Then they mixed these proteins with their cultured human fibroblasts in the presence of the QQ reagent. This reagent drew the proteins into the cells and the fibroblasts were reprogrammed into cardiac progenitor cells (CPCs). Appropriate control experiments showed that cells that were treated with QQ reagent without these proteins were not reprogrammed. Wang and Yu and they research groups also exposed the cells to three growth factors, BMP4 and activin A, to drive the cells to become heart-specific cells, and basic fibroblast growth factor to turn the cells towards a progenitor cell fate.

The next set of experiment was intended to show that their newly reprogrammed were of a cardiac nature. First, the cells clearly expressed heart-specific genes. Flk-1 and Isl-1 are genes that earmark cardiac progenitor cells, and by the eighth day of induction, the vast majority of cells expressed both these genes.

 

Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.
Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.

Second, cardiac cells can differentiate into three different cell types: heart muscle cells, blood vessels cells, and smooth muscle cells that surround the blood vessels. In mesoderm progenitors made from embryonic stem cells, inhibition of the Wnt signaling pathway can drive such cells to become heart muscle cells (see Chen, et al Nat Chem Biol 5:100–107; Willems E, et al Circ Res 109:360–364; Hudson J, et al Stem Cells Dev 21:1513–1523). However, Wang, Yu and company showed that treating the cells with a small molecule called IWR-1 that inhibits Wnt signaling drove their cells to differentiate into, not only heart muscle cells, but also endothelial (blood vessel) cells and smooth muscle cells when the cells were grown on gelatin coated dishes. When left to differentiate in culture, the cells beat synchronously and released calcium in a wave-like fashion that spread from one cell to another, suggesting that some cells were acting as pacemakers and setting the beat.

 

Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.
Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.

Then these cells were transplanted into the heart of mice that had suffered heart attacks. When compared to control hearts that received fluid, but no cells, the hearts of the animals that received protein-induced CPCs showed decreased scarring by 4 weeks after the transplantations. They also showed the growth of new heart muscle. A variety of staining experiments established that the engrafted protein-induced CPCs positive for heart muscle- and endothelial-specific cell markers. These experiments showed that transplantation of cardiac progenitor cells can not only help attenuate remodeling of the left ventricular after a heart attack, but that the protein-induced CPCs (piCPCs) can develop into cells of the cardiac lineage.

In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.
In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.

These are exciting results. It shows that direct reprogramming can occur without introducing genes into cells by means that can complicate the safety of the implanted cells. Also, because the cells are differentiated into progenitor cells, they still have the ability to proliferate and expand their numbers, which is essential for proper regeneration of a damaged tissue.

After a heart attack, the ventricle wall scars over and can become thin. However, piCPCs that have been directly reprogrammed from mature, adult cells can be used to replace dead heart muscle in a living animal.

Despite these exciting advances, further questions remain. For example, are the physiological properties of cells made from piCPCs similar enough to match the functional parameters of the heart into which they are inserting themselves? More work is necessary to answer that question. Functional equivalence is important, since a heart that does not function similarly from one end to the other can become arrhythmic, which is clinically dangerous. Further work is also required to precisely determine how well cells derived from piCPCs mature and coupling with neighboring cells. Therefore, larger animal studies and further studies in culture dishes will be necessary before this technique can come to the clinic. Nevertheless, this is a tremendous start to what will hopefully be a powerful and fruitful technique for healing damaged hearts.

Did the city of Nazareth exist at the time of the birth of Jesus?


I was discussing a recent debate that a friend attended between an atheist musician named Dan Barker and a Christian with a doctorate in New Testament Studies named Justin Bass. According to my friend’s report, the atheist questioned the existence of Nazareth, and then went on from there to assert that everything we know about Jesus is legendary. This […]

http://winteryknight.com/2015/12/24/did-the-city-of-nazareth-exist-at-the-time-of-the-birth-of-jesus/

Scientists Create Injectable Foam To Repair Degenerating Bones


Researchers in France have developed a self-setting foam that can repair defects in bones and assist growth. Eventually, this advanced biomaterial could be used to quickly regenerate bone growth and treat degenerative diseases such as osteoporosis. Sourced through Scoop.it from: gizmodo.com See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

http://pharmaceuticalintelligence.com/2015/12/24/scientists-create-injectable-foam-to-repair-degenerating-bones/

Merry Christmas to all my readers!!!


From Luke 2:1-20

1 In those days Caesar Augustus issued a decree that a census should be taken of the entire Roman world. 2 (This was the first census that took place while Quirinius was governor of Syria.) 3 And everyone went to their own town to register.

4 So Joseph also went up from the town of Nazareth in Galilee to Judea, to Bethlehem the town of David, because he belonged to the house and line of David. 5 He went there to register with Mary, who was pledged to be married to him and was expecting a child. 6 While they were there, the time came for the baby to be born, 7 and she gave birth to her firstborn, a son. She wrapped him in cloths and placed him in a manger, because there was no guest room available for them.

8 And there were shepherds living out in the fields nearby, keeping watch over their flocks at night. 9 An angel of the Lord appeared to them, and the glory of the Lord shone around them, and they were terrified. 10 But the angel said to them, “Do not be afraid. I bring you good news that will cause great joy for all the people. 11 Today in the town of David a Savior has been born to you; he is the Messiah, the Lord. 12 This will be a sign to you: You will find a baby wrapped in cloths and lying in a manger.”

13 Suddenly a great company of the heavenly host appeared with the angel, praising God and saying,

14
“Glory to God in the highest heaven,
and on earth peace to those on whom his favor rests.”

15 When the angels had left them and gone into heaven, the shepherds said to one another, “Let’s go to Bethlehem and see this thing that has happened, which the Lord has told us about.”

16 So they hurried off and found Mary and Joseph, and the baby, who was lying in the manger. 17 When they had seen him, they spread the word concerning what had been told them about this child, 18 and all who heard it were amazed at what the shepherds said to them. 19 But Mary treasured up all these things and pondered them in her heart. 20 The shepherds returned, glorifying and praising God for all the things they had heard and seen, which were just as they had been told.

Stem Cell-Based Cartilage Regeneration Could Decrease Knee and Hip Replacements


Work by Chul-Won Ha, director of the Stem Cell and Regenerative Medicine Institute at Samsung Medical Center and his colleagues illustrates the how stem cell treatments might help regrow cartilage in patients with osteoarthritis or have suffered from severe hip or knee injuries.

A 2011 report from the American Academy of Orthopedic Surgeons showed that approximately one million patients in the US alone (645,000 hips and 300,000 knees) have had joint replacements in the U.S. alone. Most joint replacements occur with few complications, artificial joints can only last for a certain period of time and some will even eventually require replacement. Also these procedures require extensive rehabilitation and are, in general, quite painful. A goal for regenerative medicine is the regenerate the cartilage that was worn away to prevent bones from eroding each other and obviate the need for artificial joint replacement procedures.

Extensive research from the past two decades from a whole host of laboratories in the United States, Europe, and Japan have shown that mesenchymal stem cells (MSCs) have the ability to make cartilage, and might even have the capability to regenerate cartilage in the joint of a living organism. MSCs have the added benefit of suppressing inflammation, which is a major contributor to the pathology of osteoporosis. Additionally, MSCs are also relatively easy to isolate from tissues and store.

“Over the past several years, we have been investigating the regeneration potential of human umbilical cord blood- derived MSCs in a hyaluronic acid (HA) hydrogel composite. This has shown remarkable results for cartilage regeneration in rat and rabbit models. In this latest study we wanted to evaluate how this same cell/HA mixture would perform in larger animals,” said Ha.

Ha collaborated with researchers from Ajou University, which is also in Seoul, and Jeju University in Jeju, Korea. Ha and his team used pigs as their model system, which is a better system than rodents for such research.

The stem cells for this project were isolated from human umbilical cord blood that was obtained from a cord blood bank. They isolated MSCs from the umbilical cord blood and grew them in culture to establish three different human Umbilical Cord Blood MSC lines. Then they pelleted the cells and mixed them with the HA solution and applied them to the damaged knee joints of pigs.

“After 12 weeks, there was no evidence of abnormal findings suggesting rejection or infection in any of the six treated pigs. The surface of the defect site in the transplanted knees was relatively smooth and had similar coloration and microscopic findings as the surrounding normal cartilage, compared to the knees of a control group of animals that received no cells. The borderline of the defect was less distinct, too,” said the study’s lead investigator, Yong-Beom Park, who is a colleague of Ha’s at the SungKyunKwan University’s Stem Cell and Regenerative Medicine Institute.

“This led us to conclude that the transplantation of hUCB-MSCs and 4 percent HA hydrogel shows superior cartilage regeneration, regardless of the species. These consistent results in animals may be a stepping stone to a human clinical trial in the future,” Dr. Ha noted.

“These cells are easy to obtain, can be stored in advance and the number of potential donors is high,” said Anthony Atala, M.D., Editor of STEM CELLS Translational Medicine and Director of the Wake Forest Institute for Regenerative Medicine. “The positive results in multiple species, including the first study of this treatment in large animals, are certainly promising for the many patients requiring treatments for worn and damaged cartilage.”

A Common Osteoporosis Drug Protects Bone Marrow Stem Cells from DNA Damage


A commonly used treatment for osteoporosis can protect stem cells in bone from the ravages of aging, according to a new study from the University of Sheffield.

Ilaria Bellantuono and her colleagues have discovered that zoledronate can extend the lifespan of bone marrow mesenchymal stem cells by reducing the degree of DNA damage experienced by these stem cells.

As stem cells age, they accumulate DNA damage, and this seems to be one of the most important mechanisms of aging. DNA damage can cause stem cells to lose their capacity to maintain tissues and repair them when those tissues are damaged. This new research from Bellantuono’s laboratory shows that zoledronate can protect mesenchymal stem cells from DNA damage, which enhances their survival and maintains their function.

According the Professor Bellantuono, “The drug enhances the repair of the damage in DNA occurring with age in stem cells in the bone. It is also likely to work in other stem cells too.”

She continued: “This drug has been shown to delay mortality in patients affected by osteoporosis but until now we didn’t know why. These findings provide an explanation as to why it may help people to live longer.

“Now we want to understand whether the drug can be used to delay or revert the aging in stem cells in older people and improve the maintenance of tissues such as the heart, the muscle and immune cells, keeping them healthier for longer.

“We want to understand whether it improves the ability of stem cells to repair those tissues after injury, such as when older patients with cancer undergo radiotherapy.”

Almost half of elderly patients over 75 years of age have three or more diseases at the same time, such as osteoporosis, diabetes, cardiovascular disease, infections, and muscle weakness. However, work like this suggests that drugs like zoledronate could be used to treat, prevent or perhaps even delay the onset of such diseases.

Dr Bellantuono added: “We are hopeful that this research will pave the way for a better cure for cancer patients and keeping older people healthier for longer by reducing the risk of developing multiple age-related diseases.”

First Stem Cell Trial for Alzheimer’s Disease Will Enroll Patients Next Year


A research group from the University of Miami Miller School of Medicine will be conducting the first clinical trial that will test the ability of stem cells to treat Alzheimer’s disease.

According the Bernard Baumel, assistant professor of neurology at the Miller School of Medicine and the principal investigator for this phase I clinical trial, said “We believe infusions of these types of stem cells have the potential to be beneficial to individuals with Alzheimer’s disease.” Because this trial is a phase 1 clinical trial, it will test the safety of this treatment strategy.

Baumel and his colleagues plan to test the safety of mesenchymal stem cells (MSCs) as a treatment for Alzheimer’s disease.  In order to acquire high-quality MSCs for this clinical trials, Dr. Baumel is collaborating with his colleague Joshua Hare, Louis Lemberg Professor of Medicine and director of the Miller School’s Interdisciplinary Stem Cell Institute (ISCI).  Dr. Hare is an expert in the use and manipulation of MSCs who has developed a life sciences company called Longeveron that isolates, characterized and stores MSCs for clinical applications.

“Stem cells are very potent anti-inflammatories,” Dr. Baumel said. “Because the amyloid plaques found in the brains of Alzheimer’s disease patients are associated with inflammation, infusions of stem cells may help to improve or stabilize that condition. Those new brain cells may then be able to replace damaged cells in Alzheimer’s patients.”

Previous work in several different laboratories has demonstrated the anti-inflammatory capacities of MSCs (Chen PM, et al J Biomed Sci. 2011; 18:49), but other laboratories have even observed that, under certain conditions, MSCs can differentiate into brain cells (Tsz Kin Ng, et al World J Stem Cells. 2014 Apr 26; 6(2): 111–119). Therefore, MSCs potentially provide a powerful one-two punch for treating Alzheimer’s disease patients.

This clinical trial is called “Allogeneic Human Mesenchymal Stem Cell Infusion Versus Placebo in Patients with Alzheimer’s Disease,” and enrollment for this trial will begin in early 2016 and continue through to 2018. Patients enrolled in the study will have their undergo cognitive function tests before and after the treatment, quality of life assessments and brain volume measurements in order to acquire some knowledge of the potential effectiveness of this cell-based treatment strategy.

Patients with mild Alzheimer’s disease but who are otherwise healthy will be encouraged to enroll in this study.

Dying Muscles Leave “Ghost Fibers” that Direct Muscle Regeneration


When muscles are injured, they die off in order to make room for the growth of replacement muscles. However, it turns out that these moribund muscle leave behind small evanescent fibers that have been called “ghost fibers.” Ghost fibers seem to be remnants of the gooey stuff that provides the substratum upon which muscle cells sit. This gooey foundation is called “extracellular matrix” or ECM. The ECM consists of acid sugars called “glycosaminoglycans,” which are given the unfortunate abbreviation of GAGs, proteins to which GAGs are attached called “proteoglycans,” and proteins that glue cells to the ECM, such as fibronectin, laminin, and collagen IV. Cells adhere to the ECM by means of receptors embedded in their cell membranes called integrins.

Extracellular matrix

Dying muscle cells leave collagen fibers in their wake and these collagen fibers constitute these so-called ghost fibers. However, these ghost fibers provide the structure into which new muscle cells are inserted. A new study by research teams at the Carnegie Institution for Science and the National Institute of Child Health and Human Development that was published in the journal Cell Stem Cell has established that ghost fibers guide new muscle cells to grow in place and ultimately heal muscle injury in laboratory mice.

Ghost Fibers
Ghost Fibers

Chen-Ming Fan at the Carnegie Institute of Washington in Baltimore, Maryland and his colleagues, in collaboration with and Jennifer Lippincott-Schwartz and her colleagues from the NIH disabled the hind limb muscles of laboratory mice by means of physical injury (laceration), or the administration of toxins. These insults to the skeletal muscles caused the injured muscle fibers to die and disintegrate. They also confirmed that as the skeletal muscle disappeared, they left networks of collagen ghost fibers in their wake.

Then, this team utilized three-dimensional, time-lapse intravital imaging to directly visualize the process of muscle regeneration in live mice. What they saw was stunning. The extracellular matrix remnants or ghost fibers left by the injured skeletal directed muscle stem/progenitor cell behavior during muscle regeneration. The two-photon imaging and second-harmonic generation microscopy employed by this team enabled them to precisely observe the muscle stem and precursor cells in individual mice orient themselves along the ghost fibers and grow new muscle tissue.

The muscle stem cells were quiescent and did not move in uninjured muscle tissue. Only when muscle cells were injured did the muscle stem cells come to life, move to the site of injury and begin the healing process. Both the cell division of these muscle stem cells and their migration were oriented along the longitudinal axes of the ghost fibers.

ImageJ=1.49m unit=inch
ImageJ=1.49m
unit=inch

If the ghost fibers were artificially reoriented, then the muscle progenitors migrated and divided in different planes and gave rise to disorganized regenerated muscle fibers.

From these results, Fan and his team concluded that “the ghost fiber (1) is a key determinant for patterning muscle stem cell behavior and (2) provides the foundation for proportional regeneration. They concluded that “ghost fibers are autonomous, architectural units necessary for proportional regeneration after tissue injury.” They continued, “This finding reinforces the need to fabricate bioengineered matrices that mimic living tissue matrices for tissue regeneration therapy.”

Scientists Grow New Diaphragm Tissue In Laboratory Animals


The diaphragm is a parachute-shaped muscle that separates the thoracic cavity from the abdominopelvic cavity and facilitates breathing. Contraction of the diaphragm increases the volume of the lungs, thus dropping the pressure in the lungs below the pressure of the surrounding air and causing air to rush into the lungs (inhalation). Relaxation of the diaphragm decreases the volume of the lungs and increases the pressure in the lungs so that it exceeds the pressure of the air, and air leaves the lungs (exhalation). The diaphragm is also important for swallowing. One in 2,500 babies are born with malformations or perforations in their diaphragms, and this condition is usually fatal.

The usual treatment for this condition involves the construction of an artificial patch that properly covers the lesion, but has no ability to grow with the infant and is not composed of contractile tissue. Therefore, it does not assist in contraction of the diaphragm to assist in breathing.

A new study might change the prospects for these newborn babies. Tissue engineering teams from laboratories in Sweden, Russia and the United States have successfully grown new diaphragm tissue in rats by applying a mixture of stem cells embedded in a 3D scaffold made from donated diaphragm tissue. Transplantation of this stem cell/diaphragm matrix concoction into rats allowed the animals to regrow new diaphragm tissue that possessed the same biological characteristics as diaphragm muscle.
The techniques designed by this study might provide the means for repairing defective diaphragms or even hearts.

Doris Taylor, who serves as the director of regenerative medicine research at the Texas Heart Institute and participated in this revolutionary study, said: “So far, attempts to grow and transplant such new tissues have been conducted in the relatively simple organs of the bladder, windpipe and esophagus. The diaphragm, with its need for constant muscle contraction and relaxation puts complex demands on any 3D scaffold. Until now, no one knew whether it would be possible to engineer.”

Paolo Macchiarini, the director of the Advanced Center for Regenerative Medicine and senior scientist at Karolinska Institutet, who also participated in this study, said: “This bioengineered muscle tissue is a truly exciting step in our journey towards regenerating whole and complex organs. You can see the muscle contracting and doing its job as well as any naturally grown tissue.”

To make their tissue engineered diaphragms, the team used diaphragm tissue that had been taken from donor rats. They stripped these diaphragms of all their cells, but maintained all the connective tissue. This removed anything in these diaphragms that might cause the immune systems of recipient animals to reject the implanted tissue, while at the same time keeping all the things that give the diaphragm its shape and form. In the laboratory, the decellularized diaphragms had lost all their elasticity. However, once these diaphragm matrices were seeded with bone marrow-derived stem cells and transplanted into recipient laboratory animals, the diaphragm scaffolds began to function as well as normal, undamaged diaphragms.

If this new technique can be successfully adapted to human patients, it could replace the damaged diaphragm tissue of the patient with tissue that would constantly contract and grow with the child. Additionally, the diaphragm could be repaired by utilizing a child’s own stem cells. As a bonus, this technique might also provide a new way to

Next, the team must test this technique on larger animals before it can be tested in a human clinical trial.

The study was published in the journal Biomaterials.

Small Molecule Supercharges Human Cardiac Stem Cells


HO-1 or heme oxygenase is an enzyme that degrades heme groups to biliverdin, iron, and carbon monoxide. It is induced in cells in response to oxidative stress. Overexpression of HO-1 can make cells more resistant to oxidative stress. The highest levels of HO-1 are found in the spleen, where old red blood cells are sequestrated and destroyed.

Mesenchymal stem cells (MSCs) from bone marrow have been genetically engineered to overexpress HO-1 survive much better when implanted into the hearts of animals that have recently suffered a heart attack (Zeng B, et Al, Biomed Sci. 2010 Oct 7;17:80; Yang JJ et al Tohoku J Exp Med. 2012;226(3):231-41). Such cells also increase the density of blood vessels in infarcted tissue, and HO-1 has been postulated to increase blood vessel production (Jang YB et al Chin Med J (Engl). 2011 Feb;124(3):401-7).

These previous experiments show that HO-1 can increase the survival and therapeutic abilities of MSCs. Can increasing the levels of HO-1 do the same for other types of stem cells?

Stuart Atkinson at the Stem Cell Portal web site has highlighted a new paper that was published in the journal Stem Cells that has examined increasing the levels of HO-1 in Cardiac Stem Cells (CSCs).

CSCs are a resident stem cell in the heart that can be isolated from heart patients during heart surgeries. Animal studies and clinical trials have shown that implantation of CSCs soon after a heart attack can produce significant increases in heart function (Bearzi C, et al. Proc Natl Acad Sci U S A 2007;104:14068-14073; Bolli R, et al Lancet. 2011 Nov 26;378(9806):1847-57). Unfortunately, the success of this clinical has been called into questioned by some problems with the data reported in this paper. However, animal studies suggest that the effectiveness of CSCs is compromised by their limited ability to survive in the heart after a heart attack (Hong KU, et al. PLoS One 2014;9:e96725). Therefore, increasing the survival of CSCs might increase their therapeutic efficacy.

Atkinson notes that the compound cobalt protoporphyrin (CoPP) can induce the expression of higher levels of HO-1 and thereby increase the resistance of the cells to oxidative stress and augment cell survival. Therefore, Robert Bolli from the University of Louisville, Kentucky and his colleagues, in collaboration with researchers from the Albany Medical College have treated CSCs with CoPP and these tested their ability to heal the heart after a heart attack.

Bolli and others isolated human CSCs from patients undergoing CABG (cardiac artery bypass graft) surgery, and grew them in culture to beef up the numbers of cells. After a short time in culture, the CSCs were incubated with CoPP for 12 hours. Then Bolli and his team transplanted these human CSCs that were also labeled with green fluorescent protein (GFP) into the hearts of mice that had suffered rather massive heart attacks and had undergone 35 days of reperfusion. The GFP allowed Bolli and others to detect the presence of the implanted CSCs in the rodent heart tissue.

When these hearts of these mice were examined one and five weeks after CSC transplantation, the CoPP-treated CSCs showed substantially higher levels of survival in the mouse hearts. The other two groups of mice included those transplanted with non-pretreated CSCs, and mice treated with the culture medium used to grow the CSCs, and the pretreated CSCs survival significantly better than the non-pretreated CSCs.

CoPP pretreatment seems to augment cell survival, but do the surviving cells increase heart function? Bolli and others used echocardiogram to measure heart function, and echocardiographic assessment 5 weeks after CSC transplantation showed that the CoPP-preconditioned CSCs elicited greater improvement in remodeling of the left ventricle. Additionally, the hearts of the animals that received CoPP-pretreated CSCs showed improved movement of the walls of the heart during its pumping cycle, and better overall performance of the heart in general. Both pretreated and the non-pretreated CSCs, but not CSC culture growth medium shrank the amount of scar tissue in the heart and grew new heart tissue. However, The CoPP-pretreated CSCs were obviously superior to the non-pretreated CSCs at increasing the mass of heart muscle (see here for pictures).

These experiments might very well unravel a burning controversy surrounding CSCs. Bolli’s experiment show that can definitely grow new heart muscle. However, the bulk of the experiments with CSCs strongly suggest that these cells improve heart function by secreting pro-healing molecules without directly contributing to the regrowth of heart muscle. These papers probably observed the effects of CSCs that were transplanted into the heart, but did not survive very long. Bolli and his colleagues, on the other hand, were able to implant CSCs and survived for a much longer time in the hearts. Incidentally, Bolli and his team showed that the implanted CSCs expressed heart muscle-specific genes, which corroborated that these cells were differentiating into heart muscle cells, even though the proportion of cells that formed new heart muscle was relatively small.

In summary, CoPP pretreatment of cell seems to be feasible, safe, and effective as a means to improve CSC-based therapy. Even though It is likely that paracrine mechanisms are essential for CSC-based healing, the ability of CSCs to differentiate into heart muscle cells also seems to be an essential part of the means by which CSCs heal the heart after a heart attack. Thus more work is certainly warranted, but this is a fine start to what might be a simple, but effective way to increase the effectiveness of our own CSCs.

Accelerated Reprogramming and Gene Editing Protocol Can Make Fixed Cells Much Faster


Sara Howden and her colleagues at the Morgridge Institute for Research and the Murdoch Children’s Research Institute in Australia have devised a protocol that can significantly decrease the time involved in reprogramming mature adult cells while genetically repairing them at the same time. Such an advance is essential for making future therapies possible.

Howden and others demonstrated that genetically repaired cells can be derived from patient skin cells in as little as two weeks. This is much shorter than the multistep approaches that take more than three months.

How were they able to shorten the time necessary to do this? They combined two integral steps in the procedure. Adult cells were reprogrammed to an embryonic stem cell-like state in order to be differentiated into the cells that we want. Secondly, the cells must undergo gene editing in order to correct the disease-causing mutation.

By in this new protocol developed by Howden and her colleagues, they combined the reprogramming and gene editing steps.

To test their new protocol, Howden and her team used cells isolated from a patient with an inherited retinal degeneration disorder, and an infant with severe immunodeficiency. In both cases, the team not only derived induced pluripotent stem cell lines from the adult cells of these patients, but they were also able to repair the genetic lesion that causes the genetic disease.

This protocol might advance transplant medicine by making gene-correction therapies available to patients in a much timelier fashion and at lower cost.

Presently, making induced pluripotent stem cell lines from a patient’s cells, genetically repairing those cells, expanding them, differentiating them, and then isolating the right cells from transplantation, while checking the cells all along the way and properly characterizing them for safety reasons would take too long and cost too much.

With this new approach, however, Howden and others used the CRISPR/Cas9 technology to edit the damaged genes while reprogramming the cells, greatly reducing the time required to make the cells for transplantation.

Faster reprogramming also decreases the amount of time the cells remain in culture, which minimizes the risks of gene instability or epigenetic changes that can sometimes occur when culturing cells outside the human body.

Howden’s next goal is to adapt her protocol to work with blood cells so that blood samples rather than skin biopsies can be used to secure the cells for reprogramming/gene editing procedure. Blood cells also do not require the expansion that skin cells require, which would even further shorten the time needed to make the desired cell types.

The accelerated pace of the reprogramming procedure could make a genuine difference in those cases where medical interventions are required in as little time as possible. For example, children born with severe combined immunodeficiency usually die within the first few years of life from massive infections.

Howden cautioned, however, that she and her team must first derive a long-term source of blood cells from pluripotent stem cells before such treatments are viable and demonstrate the safety of such treatments as well.

See Stem Cell Reports, 2015: DOI: 10.1016/j.stemcr.2015.10.009.

How Stem Cell Therapy Protects Bone In Lupus


Systemic Lupus Erythematosis, otherwise known as lupus, is an autoimmune disease cause your own immune system attacking various cells and tissues in your body. Lupus patients can suffer from fatigue, joint pain and selling and show a marked increased risk or osteoporosis.

Clinical trials have established that infusions of mesenchymal stem cells (MSCs) can significantly improve the condition of lupus patients, but exactly why these cells help these patients is not completely clear. Certainly suppression of inflammation is probably part of the mechanism by which these cells help lupus patients, but how do these cells improve the bone health of lupus patients?

Songtao Shi and his team at the University of Pennsylvania have used an animal model of lupus to investigate this very question. In their hands, transplanted MSCs improve the function of bone marrow stem cells by providing a source of the FAS protein. FAS stimulates bone marrow stem cell function by means of a multi-step, epigenetic mechanism.

This work by Shi and his colleagues has implications for other cell-based treatment strategies for not only lupus, but other diseases as well.

“When we used transplanted stem cells for these diseases, we didn’t know exactly what they were doing, but saw that they were effective,” said Shi. “Now we’ve seen in a model of lupus that bone-forming mesenchymal stem cell function was rescued by a mechanism that was totally unexpected.”

In earlier work, Shi and his group showed that mesenchymal stem cell infusions can be used to treat various autoimmune diseases in particular animals models. While these were certainly highly desirable results, no one could fully understand why these cells worked as well as they did. Shi began to suspect that some sort of epigenetic mechanism was at work since the infused MSCs seemed to permanently recalibrate the gene expression patterns in cells.

In order to test this possibility, Shi and others found that lupus mice had a malfunctioning FAS protein that prevented their bone marrow MSCs from releasing pro-bone molecules that are integral for bone maintenance and deposition.

A deficiency for the FAS protein prevents bone marrow stem cells from releasing a microRNA called miR-29b.  The failure to release miR-29b causes its concentrations to increase inside the cells.  miR-29b can down-regulate an enzyme called DNA methyltransferase 1 (Dnmt1), and the buildup of miR-29b inhibits Dnmt1, which causes decreased methylation of the Notch1 promoter and activation of Notch signaling.  Methylation of the promoters of genes tends to shut down gene expression, and the lack of methylation of the Notch promoter increases Notch gene expression, activating Notch signaling.  Unfortunately, increased Notch signaling impaired the differentiation of bone marrow stem cells into bone-making cells.  Transplantation of MSCs brings FAS protein to the bone marrow stem cells by means of exosomes secreted by the MSCs.  The FAS protein in the MSC-provided exosomes reduce intracellular levels of miR-29b, which leads to higher levels of Dnmt1.  Dnmt1 methylates the Notch1 promoter, thus shutting down the expression of the Notch gene, and restoring bone-specific differentiation.

Shi and others are presently investigating if this FAS-dependent process is also at work in other autoimmune diseases.  If so, then stem cell treatments might convey similar bone-specific benefits.

Faster Bone Regeneration With a Little Wnt


Nick Evans and his colleagues at the University of Southampton, UK have discovered that transient stimulation of the Wnt signaling pathway in bone marrow stem cells expands them and enhances their bone-making ability. This finding has led to an intense search for drugs that can stimulate the Wnt pathway in order to stimulate bone formation in wounded patients.

The Wnt pathway is a highly conserved pathway found in sponges, starfish, sharks, and people. Wnt signaling controls pattern formation during development, and the growth of stem cells during healing.

When it comes to healing, bone fractures represent a sizeable societal problem, particularly among the aged. While most fractures heal on their own, approximately 10 percent of all fractures take over six months to heal or never heal at all. In the worse cases, fracture patients can require several surgeries or might need amputation in desperate cases.

According the Evans, he and his research group are screening a wide range of chemicals to determine if they stimulate Wnt signaling. If such chemicals prove safe to use in laboratory animals, then they might become clinical tools to help stimulate bone formation and healing in patients with recalcitrant fractures.

Research from Evans’ group has shown that transient stimulation of the Wnt signaling pathway in isolated bone marrow cells increases the number of bone-making progenitor cells. However, if the Wnt pathway is activated for too long a time period, this regenerative effect is lost or even reversed. Hence the need to develop treatments that deliver small molecules that stimulate Wnt signaling in bone marrow cells for a specified period of time and in a targeted fashion.

Evans and his group have used nanoparticles loaded with Wnt proteins to do exactly that. The feasibility of this technology and its effectiveness requires further work, but the promise is there and the idea is more than a little intriguing.

Exosomes from Placental Mesenchymal Stem Cells Confer Plasticity to Fibroblasts


Journal of Cellular Biochemistry
DOI: 10.1002/jcb.25459

Cells have the ability to internalize small portion of their membranes into small vesicles called endosomes. Once inside the cell, these endosomes can then internalize bits of their own membranes to form a kind of vesicle-within-a-vesicle structure called a “multivesicular endosomes” or MVEs. Cells can load up these MVEs with various proteins and RNAs and lipids, and then secrete them into their environment. Once released, these former MVEs become known as “extracellular vesicles” or EVs, and the smallest of the EVs are known as exosomes.

Exosome_Basics

Neighboring cells can take up exosomes released by nearby or far-flung cells and the proteins and RNAs delivered by the exosomes can elicit profound changes in cell behavior. Therefore, exosomes act as agents of cell-cell communication. Detection of exosomes from cancer cells into blood, or other bodily fluids can provide diagnostic insights into the diagnosis of cancers. Also exosomes from stem cells can augment tissue healing and repair (see here).

Exosomes from mesenchymal stem cells (MSCs) are released into the culture medium when MSCs are grown in the laboratory. Exosome-containing culture medium is often referred to as “conditioned medium” and in this case it is conditioned medium from MSCs, which we will refer to as MSC-CM. MSC-CM has been reported to enhance wound healing in several different experiments.

In a new study by Motohiro Komaki and his colleagues at the Tokyo Medical and Dental University, in Tokyo, Japan has examined the effects of exosomes from human placenta MSCs in a controlled culture system to better characterize the biological roles of these exosomes.

In this study, placental MSCs were from donated human placenta from newborn babies by means of enzymatic digestion. Exosomes were prepared from these placental MSCs (PlaMSCs) by growing the cells in culture and then subjecting the conditioned culture medium to ultracentrifugation. Exosomes, fortunately, are relatively easy to isolate and can be frozen and stored quite effectively without loss of function.

These isolated PlaMSC exosomes were then given to cultured human dermal fibroblasts. Changes in gene expression were then ascertained by real-time reverse transcriptase PCR analysis (real-time PCR).

After incubating fibroblasts with exosomes from PlaMSCs, the expression of stemness-related genes, such as OCT4 and NANOG were significantly increased. These exosomes also stimulated the differentiation of these fibroblasts into bone cells and fat cells when the cells were subjected to bone and fat induction media. The fibroblasts expressed bone-specific and fat-specific genes and enzymes.

Thus, these experiments showed that exosomes from PlaMSCs increased the expression of the stemness genes OCT4 and NANOG in fibroblasts, which normally do not express these genes. Consequently, these exosomes influence ability of fibroblasts to differentiate in both bone and fat cells. This work demonstrates a new feature of MSCs and their exosomes, and suggests new possible clinical applications for MSC exosomes.

Quad Technologies Introduces New Cell Separation Technologies


Quad Technologies in Woburn, Massachusetts has developed a new cell separation system that might revolutionize cell purification.

Adopting cell technologies for clinical applications requires the manipulation of large numbers of cells that contain specific cell types that must be isolated from the rest of the cells. The isolation and purification of large numbers of cells is integral to cell-based therapies, but such procedures are often, expensive, laborious, tedious, and sometimes inefficient.

After securing proper financing, Quad Technologies Inc., launched its MagCloudz Streptavadin Cell Separation Kit, which is a commercially available product that incorporates QuickGel technology.

QuickGel is a hydrogel that is compatible with cells and is non-cytotoxic. This gel can be adapted for use with antibodies or types of tools that are used to isolate different cell populations. When antibodies or other proteins are applied to cells so that they can bind to the surfaces of specific cell types and facilitate purification of that cell population, a major limitation is the antibody finding all the cells in a culture. My immobilizing the cells if a gel matrix, the antibody has a much easier job of finding its target cells. Such gels, therefore, can greatly increase the binding of antibodies or some other purification protein. The gel is also dissolvable and easily removed without damaging targeted cells. QuickGel can greatly increase the purification efficiency of cells for therapeutic purposes.

The MagCloudz kit uses magnetic beads that bind specifically to cells that can be used to purify that cell population efficiently and quickly. The beads can then be easily released from the cells without compromising the viability or quality of the cells.

These enhanced cell purification technologies will deliver breakthrough therapies for stem cell clinics.

Inhibition of Gli1 Enhances Remyelination Abilities of Endogenous Stem Cell Populations


Nerve cells, otherwise known as neurons, have long extensions called axons. Nerve impulses travel down these axons, away from the cell body towards another neuron that is connected to the neuron. The axons of some neurons are insulated with a special substance called myelin the layer of myelin that surrounds the axon. This “myelin sheath” acts as a protective covering composed of protein and lipids.

myelin_sheath

Axons can vary in length from anywhere to 1 millimeter or less to 1 meter. Sometimes, axons are bundled together to form nerves that transmit electrical nerve impulses across the body.

While myelin protects and insulates axons, it also enhances the speed at which nerve impulses are transmitted through the axon. Axons without myelin sheaths conduct nerve impulses continuously throughout the axon. However, myelinated axons have small, uncovered gaps in the myelin sheath called nodes of Ranvier. Myelinated axons can only conduct nerve impulse at the nodes of Ranvier. Consequently, the nerve impulse jumps from node to node, greatly increasing the speed of nerve impulse conduction.

nodes_of_ranvier

If myelin is damaged, the speed of nerve impulse transmission slows substantially. Multiple sclerosis is one example of a disease that causes systematic loss of the myelin sheath. Inflammatory demyelinating diseases also cause progressive damage and loss of the myelin sheath. Regenerating the myelin sheath in these patients is one of the goals of regenerative medicine.

A good deal of data tells us that endogenous remyelination does occur. Unfortunately, this process is overwhelmed by the degree of demyelination in these diseases. A stem cell population called the parenchymal oligodendrocyte progenitor cells and endogenous adult neural stem cells in the brain are known to remyelinate demyelinated axons.

The Salzer laboratory at the New York Neuroscience Institute examined the ability of a specific adult neural stem cell population to remyelinate axons. These stem cells expressed the transcription factor Gli1.

Salzer and his team showed that this subventricular zone-specific group of neural stem cells were efficiently recruited to demyelinated portions of the brain. This same neural stem cell population was never observed entering healthy axon tracts. This finding shows that these cells seem to specialize in making new myelin sheaths for damaged axon tracts.

Since these neural stem cells expressed Gli1, and since there are drugs that can inhibit Gli1 activity, Salzer’s group wanted to show that Gli1 was a necessary factor for neural stem cell activity. Surprisingly, differentiation of these neural stem cells into oligodendrocytes (which make myelin and remyelinate axons) is significantly enhanced by inhibition of Gli1.

A specific signaling pathway called the hedgehog pathway is known to activate Gli1 and other members of the Gli gene family. However, when the hedgehog pathway in these neural stem cells was completely inhibited, it did not have the same effect and Gli1 inhibition. This suggests that Gli1 is doing more than responding to the hedgehog pathway in these neural stem cells.

Salzer and his colleagues showed that Gli1 inhibition improved myelin deposition in an animal model of experimental autoimmune encephalomyelitis; an inflammatory demyelination disease. Thus, inhibition of Gli1 activity in this preclinical model system increase regeneration of the myelin sheath in demyelinated neurons.

This work elegantly showed that endogenous neural stem cells that can remyelinate axons are present and can be activated by inhibiting Gli1. Furthermore, this activation will nicely enhance the therapeutic capacity of these endogenous cells. This potentially identifies a new therapeutic avenue for the treatment of demyelinating disorders.

This work was published in Nature. 2015 Oct 15;526(7573):448-52. doi: 10.1038/nature14957.