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.”

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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/