The Isolation of Dental Stem Cell Lines and How They Repair Teeth


Research teams at INSERM and Paris Descartes University have isolated dental stem cell lines and detailed the natural mechanism by which such cells repair lesions in teeth. This discovery could provide the foundation for therapeutic strategies that mobilize resident dental stem cells and amplify their intrinsic capacity for repair.

Teeth are mineralized organs that are anchored within the gums by roots. The outermost layer of the tooth is the enamel, which is one of the hardest substances in the body. Beneath the enamel is the dentine (sometimes known as the ivory), and the dentin (dentine if you are British) is filled with microscopic tubes that radiate from the inner pulp to the surface. These dentinal tubules contain cells called odontoblasts, which are the cells that made the dentin in the first place, and dentinal fluid (contains a mixture of albumin, transferrin, tenascin and proteoglycans). The odontoblasts maintain the dentin, but as we age, the dentin tubules calcify. Dentin is a yellowish, bone-like matrix that is porous. It is softer than enamel and decays more rapidly and is subject to severe cavities if not properly treated. Beneath the dentin is the pulp, which contains blood vessels and nerves and also houses a resident stem cell population.

Tooth Anatomy

When a dental lesion appears, the dormant stem cells in the pulp awaken and try to repair the tooth, but the means by which these cells do this is unknown.

To address these gaps in our knowledge, researchers from INSERM (French Institute of Health and Medical Research or Institut national de la santé et de la recherche), and the Paris Descartes University have extracted and isolated dental stem cells from the pulp of mouse molars and analyzed them further.

In the midst of their characterization of these cells, the French teams discovered that these dental pulp stem cells possess five different cell surface receptors for the neurotransmitters dopamine and serotonin. The present of these receptors suggested that the response of dental pulp stem cells to tooth injury was mediated by these neurotransmitters. Blood platelets, for example, are activated by binding serotonin and dopamine. Could these dental be activated by similar means?

The first set of experiments examined tooth repair in mice that lacked platelets that produced serotonin or dopamine. Such mice failed to repair tooth lesions, suggesting that serotonin and dopamine are important to inducing stem cell-mediated tooth repair.

Next, these laboratories characterized these five receptors and found that four of them were intimately involved in tooth repair; knocking out any one of the would abrogate tooth repair responses.

“In stem cell research, it is unusual to be simultaneously able to isolate cell lines, identify the markers that allow them to be recognized (here the receptors), discover the signal that recruits them (serotonin and dopamine), and discover the source of that signal (blood platelets). In this work, we have been able, unexpectedly, to explore the entire mechanism,” said Odile Kellermann, the principal author of this work.

Dentists use pulp capping materials like calcium hydroxide and tricalcium phosphate-based biomaterials to repair the tooth and fill lesions. These new findings, however, could produce new therapeutic strategies aimed at mobilizing the resident dental pulp stem cells to magnify the natural reparative capacity of teeth without the use of replacement materials.

One Type of Lung Cell Can Regenerate Another


A collaboration between the Perelman School of Medicine at the University of Pennsylvania and Duke University has found that lung tissue has a much great ability to regenerate than previously thought.

Lungs contain thousands of tiny clusters of sacs called alveoli. Gas-exchange between the air and our blood stream occurs across the thin lining of the alveoli, which are lined with extensive networks for diminutive blood vessels called capillaries. The cells that form the paper-thin lining of the alveoli are called type 1 cells. Within the alveoli are cells called type 2 cells, which secrete surfactant; a soapy substance that prevents the alveoli from collapsing upon themselves when we exhale. Some premature babies do not make enough surfactant and must be treated with surfactant to help them breathe.

Work in mice demonstrated that both type 1 and type 2 cells descend from a common embryonic precursor during lung development. When mice had bits of their lungs removed, labeling studies established that the newly re-established type 2 cells were made from type 1 cells and that some of the newly made type 1 cells were formed from type 2 cells. These results were confirmed by cell culture experiments that grew single type 1 or type 2 lung cells in culture; in both cases, the cultures gave rise to mixed cultures consisting of both type 1 and type 2 lung cells. These data demonstrate that type 1 lung cells can give rise to type 2 lung cells and visa versa.

Previously, the Duke University term had demonstrated that type 2 lung cells in mice not only produce surfactant, but also function as progenitors for other lung cells in adult mice. This shows that type 2 lung cells can definitely differentiate into type 1 lung cells. However, there was no evidence that type 1 lung cells could give rise to other types of lung cells.

In this present work, however, lung injury in mice stimulated the type 1 cells to divide and differentiate into type 2 cells over a period of three weeks while the lung regenerated. According to Jonathan Epstein from the University of Pennsylvania, It’s as if the lung cells can regenerate from one another as needed to repair missing tissue, suggesting that there is much more flexibility in the system than we have previously appreciated. These aren’t classic stem cells that we see regenerating the lung. They are mature lung cells that awaken in response to injury. We want to learn how the lung regenerates so that we can stimulate this process in situations where it is insufficient, such as in patients with COPD (chronic obstructive pulmonary disease).”

This is one of the first studies to demonstrate that mature cells that were thought to be completely at the end of their growth and differentiation capabilities can revert to an earlier state under the right conditions without the use of transcription factors, but by responding to damage.

These two research teams are also applying the approaches outlined in this publication to cells from other tissues, such as the intestine and skin, in order to study the mechanisms of cell maintenance and differentiation, and then relate these same mechanisms back to the heart. They also hope to apply these findings in clinical settings for patients who suffer from idiopathic pulmonary fibrosis, acute respiratory distress syndrome and other such conditions where the alveoli cannot supply sufficient amounts of oxygen to the blood.

Allogeneic Stem Cell Transplantation [9.2]


Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Allogeneic Stem Cell Transplantation

Larry H. Bernstein, MD, FCAP, Writer and Curator

http://pharmaceuticalintelligence.com/2015/lhbern/Allogeneic_Stem_Cell_Transplantation

9.2 Allogeneic Stem Cell Transplantation

9.2.1 Allogeneic Stem Cell Treatment

http://www.lls.org/treatment/types-of-treatment/stem-cell-transplantation/allogeneic-stem-cell-transplantation

Allogeneic stem cell transplantation involves transferring the stem cells from a healthy person (the donor) to your body after high-intensity chemotherapy or radiation.

Allogeneic stem cell transplantation is used to cure some patients who:

  • Are at high risk of relapse
  • Don’t respond fully to treatment
  • Relapse after prior successful treatment

Allogeneic stem cell transplantation can be a high-risk procedure. The high-conditioning regimens are meant to severely or completely impair your ability to make stem cells and you will likely experience side effects during the days you receive high-dose conditioning radiation or chemotherapy. The goals of high-conditioning therapy are to:

treat the remaining cancer cells intensively, thereby making a cancer recurrence less likely
inactivate the immune system to reduce the chance of stem cell graft rejection
enable…

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Stem Cells Inc Spinal Cord Injury Trial Shows Sustained Improvements in Sensory Function


A cellular therapeutic company known as Stem Cells, Incorporated has been carrying out a Phase I/II clinical trial that was specifically designed to assess both safety and preliminary efficacy of their proprietary HuCNS-SC cells as a treatment for chronic spinal cord injury. Recently, Dr. Armin Curt, the principal investigator of this clinical trial, presented a summary of the safety and preliminary efficacy data from this Phase I/II study at the 4th Joint International Spinal Cord Society (ISCoS) and American Spinal Injury Association (ASIA) meeting which was held in Montreal, Canada.

Spinal cord injury patients are classified by a system that was developed by the American Spinal Injury Association (ASIA) and uses grades A through E on the American Spinal Injury Association Impairment Scale (AIS) to indicate the severity of the spinal cord injury. AIS Grade A injuries consist of a loss of all spinal cord function (sensation and movement) below the level of injury is lost. This is known as a complete injury. All the other AIS grades are considered incomplete. Patients with Grade B injuries have some sensation below the level of injury, but there is no movement below the injury.. In patients with AIS Grade C injuries, there is both sensation and movement, but most of the muscles below the injury cannot function against resistance and that includes gravity. Those with AIS Grade D spinal cord injuries have some sensation and movement, but more than half of the muscles below the injury can function against resistance. Finally those with AIS Grade E injuries have both normal sensation and movement, but there may be other signs of injury, for example, pain.

For this trial, Stem Cell Inc enrolled 12 subjects who had suffered from a severe spinal cord injury at the thoracic or chest level (T2-T11); seven AIS A and 5 AIS B patients.. In order to qualify for this study, all patients had to be classified as either AIS A or B and a minimum of 3 months from injury.

The trial involved internationally prominent medical centers for spinal cord injury and rehabilitation, and associated principal investigators; Dr. Armin Curt at the University of Zurich and Balgrist University Hospital, Dr. Steve Casha at the University of Calgary, and Dr. Michael Fehlings at the University of Toronto.

All subjects in this trial received HuCNS-SC cells by means of direct transplantation into the spinal cord and they were also treated, temporarily, with immunosuppressive drugs to prevent the immune system from rejecting the implanted cells. Patients were regularly evaluated for safety of the treatment protocol, and to determine if patients showed any change in neurological function. To determine this, patients were given a standard battery of movement and sensory tests before the surgery and at routine intervals after the procedure. Thus all patients were simultaneously enrolled in a safety evaluation and separate evaluation that tested the efficacy of the procedure as well.

In the safety analyses of these subjects, all the data demonstrated that the surgical transplantation technique and cell dose were safe and well tolerated by all patients. HuCNS-SC cells were injected directly into the spinal cord both above and below the level of injury and none of the patients in sequential examinations over the course of twelve months showed any abnormal changes in spinal cord function associated with the transplantation technique. Additionally, there were no adverse events that could be attributed to the HuCNS-SC cells.

Analyses of the functional data after twelve-months revealed sustained improvements in sensory function that emerged consistently around three months after transplantation and persisted until the end of the study. These gains in sensory function involved multiple sensory pathways and were observed more frequently in the patients with less severe spinal cord injuries. Three of the seven AIS A patients and four of the five AIS B patients showed signs of positive sensory gains. Two patients in the study progressed from AIS A, to the lesser degree of injury grade, AIS B.

“It has been a privilege to be a part of the first study to test the potential of neural stem cell transplantation in thoracic spinal cord injury,” said Dr. Armin Curt, Professor and Chairman of the Spinal Cord Injury Center at Balgrist University Hospital, University of Zurich. “The gains we have detected indicate that areas of sensory function have returned in more than half the patients. Such gains are unlikely to have occurred spontaneously given the average time from injury. This patient population represents a form of spinal cord injury that has historically defied responses to experimental therapies, and the measurable gains we have found strongly argue for a biological result of the transplanted cells. These gains are exciting evidence that we are on the right track for developing this approach for spinal cord injury. This early outcome in thoracic injury firmly supports testing in cervical spinal cord injury.”

Stephen Huhn, M.D., FACS, FAAP, Vice President, Clinical Research and CMO at StemCells, Inc., said, “This research program has the potential to revolutionize the therapeutic paradigm for spinal cord injury patients. The clinical gains observed in this first study are a great beginning. We found evidence of sensory gains in multiple segments of the injured thoracic spinal cord across multiple patients. Our primary focus in this study for spinal cord injury was to evaluate safety and also to look for even small signs of an effect that went beyond the possibility of spontaneous recovery. We are obviously very pleased that the pattern of sensory gains observed in this study are both durable and meaningful, and indicate that the transplantation has impacted the function of damaged neural pathways in the cord. The Company’s development program has now advanced to a Phase II controlled study in cervical spinal cord injury where the corollary of sensory improvements in thoracic spinal cord injury could well be improved motor function in the upper extremities of patients with cervical spinal cord injuries.”

CAR T-Cell Therapy Surpasses 90% Complete Remission Rate in Pediatric ALL


The chimeric antigen receptor (CAR) T-cell therapy JCAR017 elicited a 91% complete remission rate in pediatric patients with relapsed/refractory acute lymphoblastic leukemia (ALL), according to results from a phase I trial presented at the 2015 AACR Annual Meeting.

In the treated patients, complete remissions were observed in 20 of 22 patients, as ascertained by flow cytometry.  Complete remissions were observed with all applied doses of JCAR017 and in patients who had been treated already with CD19-targeted therapies.  Severe neurotoxicity and/or severe “cytokine release syndrome” was observed in 8 patients. In total, 4 patients have relapsed—only one of which had CD19-positive disease.

“The 91% remission rate in this phase I study of JCAR017 is highly encouraging, particularly when considering these pediatric patients failed to respond to standard treatments,” Michael Jensen, MD, said in a statement. “Based on these results we are eager to advance this study, and to continue advancing the use of cell therapies to change how we treat cancer and provide patients the opportunity for better treatment options.”

Jensen serves as the director of the Ben Towne Center for Childhood Cancer Research at Seattle Children’s Research Institute, and is also the scientific co-founder of Juno Therapeutics, which is the company that is developing JCAR017.  JCAR017 is being evaluated in an ongoing phase I/II study for pediatric and young adult patients with relapsed/refractory CD19-positive leukemia at the Seattle Children’s Hospital.

This  study intends to enroll 80 patients.  The phase I portion enrolled patients who had undergone an allogeneic hematopoietic cell transplant, but the second phase of the study is open to patients, regardless of prior transplant status (NCT02028455).

“Given the impressive clinical results with this defined cell product candidate, we are encouraged to begin testing of JCAR017 in adult patients with B cell malignancies, including non-Hodgkin lymphoma, later this year,” Hans Bishop, chief executive officer of Juno Therapeutics, said in a statement.

Juno also has three other CAR and T cell receptor therapies that are under evaluation in clinical trials.  The furthest along is JCAR015, which received a breakthrough therapy designation from the FDA as a treatment for patients with relapsed or refractory B-cell ALL in November 2014.  In one trial, JCAR015 is being used to treat precursor B cell ALL, which is the condition for which JCAR015 was awarded its orphan drug designation (NCT01840566).  In a second trial, patients with relapsed/refractory aggressive B cell non-Hodgkin lymphoma (NHL) are being treated with high dose therapy and autologous stem cell transplantation followed by infusion of JCAR015 (NCT01044069).

There are plans for future clinical trials to explore Juno’s CAR T cell therapies in combination with immune checkpoint inhibitors. Recently, Juno and MedImmune, the biologics research and development arm of AstraZeneca, announced an agreement focused on the clinical development of combination strategies.  A jointly-funded phase Ib study will explore one of Juno’s CD19-directed CAR T cell therapies in combination with the PD-L1 inhibitor MEDI4736 as a treatment for patients with NHL.  It is expected to begin later this year.

“We believe combination strategies such as this will help us better understand the full potential of our engineered T cell platform in both hematological and solid tumor settings,” Mark W. Frohlich, MD, executive vice president, Research & Development, Juno Therapeutics, said in a statement.

MEDI4736 is currently under evaluated in phase III clinical trials that are testing MEDI4736 alone and in combination with the CTLA-4 antibody tremelimumab in patients with a variety of non-small cell lung cancers and in patients with head and neck cancer who have failed prior chemotherapy.

Competition in the field of immuno-oncology has resulted in collaborations between several pharmaceutical companies, outside of the Juno deal. This is evident in the field of CAR-modified T-cell therapy, where collaborations exist between Novartis and the University of Pennsylvania (CTL019) and between Kite Pharma and the NCI (KTE-C19).

In the pediatric oncology space, results from the breakthrough therapy CTL019 were presented at the 2014 ASH Annual Meeting and demonstrated similar findings to those announced for JCAR017. In this phase I trial of 39 pediatric patients with relapsed/refractory ALL, CTL019 demonstrated a 92% complete remission rate. In total, 85% of patients who achieve a complete remission tested MRD-negative by flow cytometry.

Making Platelets in the Culture Dish


Bone marrow-based cells known as megakaryocytes are rather uncommon in bone marrow, but these cells are very important for the health and daily operation of the human body. Megakaryocytes, you see, produce platelets, which are critical to clotting broken blood vessels and wound healing. Generating megakaryocytes in cell culture has proven to be rather difficult, but induced pluripotent stem cells might provide a way to make megakaryocytes in culture.

Megakaryocyte
Megakaryocyte

Platelets_large

The differentiation of induced pluripotent stem cells (iPSCs) into megakaryocytes could potentially create a renewable cell source of platelets for treating patient with “thrombocytopenia,” which is a deficiency of platelets. Zack Wang and his colleagues from Johns Hopkins University in Baltimore, Maryland have developed a protocol to make megakaryocytes in culture from iPSCs. However, more than that, Wang and his co-workers wanted to make patient-specific platelets in culture without using any animal products and with compounds that were approved by the US Food and Drug Association. Such a protocol would demonstrate that using such cells in human patients is feasible and safe.

Wang and his colleagues developed an efficient system that generated megakaryocytes from human iPSCs without the use of animal feeder cells and without animal products (known as xeno-free condition). Several crucial reagents necessary to differentiate iPSCs into megakaryocytes into were replaced with Food and Drug Administration-approved pharmacological reagents that included romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). Wang and his group used their method to induce megakaryocytes generation from human iPSCs derived from 23 individuals in two steps: 1) generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and 2) generation and expansion of CD41+CD42a+ megakaryocytes from HPCs for an additional 5 days. After 19 days, Wang and his group observed abundant CD41+CD42a+ megakaryocytes that also expressed the megakaryocyte-specific cell-surface proteins CD42b and CD61. These cells were also polyploid, which means that they had multiple copies of each chromosome rather than just 2 copies (≥16% of derived cells with DNA contents >4N). Gene expression studies showed that megakaryocytic-related genes were highly expressed in their cultured megakaryocytes.

Characterization of human induced pluripotent stem cell-derived MKs. (A): Representative images of CFU-MK colonies taken from D14 (upper) and D19 (lower) suspension cells. All the colonies containing at least 50 CD41+ cells were considered CFU-MKs. (B): The number of CFU-MK colonies from 1.5 × 105 isolated CD34+ cells on days 14 and 19. The colonies were counted after 12 days of culture from one 35-mm dish. Mean ± SD; n = 3; ∗∗, p < .01. (C): DNA content analysis by flow cytometry on day 19. Left: The whole population stained by propidium iodide. Right: Double staining using CD41-APC and DAPI, gated on CD41+ population. (D): Wright-Giemsa staining of the suspension cells on day 19. Scale bars = 100 μm. Abbreviations: CFU, colony-forming unit; D, day; MKs, megakaryocytes.
Characterization of human induced pluripotent stem cell-derived MKs. (A): Representative images of CFU-MK colonies taken from D14 (upper) and D19 (lower) suspension cells. All the colonies containing at least 50 CD41+ cells were considered CFU-MKs. (B): The number of CFU-MK colonies from 1.5 × 105 isolated CD34+ cells on days 14 and 19. The colonies were counted after 12 days of culture from one 35-mm dish. Mean ± SD; n = 3; ∗∗, p < .01. (C): DNA content analysis by flow cytometry on day 19. Left: The whole population stained by propidium iodide. Right: Double staining using CD41-APC and DAPI, gated on CD41+ population. (D): Wright-Giemsa staining of the suspension cells on day 19. Scale bars = 100 μm. Abbreviations: CFU, colony-forming unit; D, day; MKs, megakaryocytes.

This protocol could be used to further understand the medical conditions that lead to thrombocytopenia. Deeper understanding of these medical conditions will hopefully lead to better treatments of them. Also, Wang’s protocol may lead to the generation of large numbers of platelets in culture that could then be given to patients who need them.

Using Peptides to Reset a Diseased Cell


Researchers at the University of California, San Diego School of Medicine have published a series of proof-of-concept experiments that demonstrate the ability to direct medically relevant cell behaviors by artificially manipulating a central hub in cell communication networks. The manipulation of this communication node, which was reported in the journal Proceedings of the National Academy of Sciences, makes it possible to reprogram major parts of a cell’s signaling network instead of targeting only a single receptor or cell signaling pathway.

This discovery could have tremendous clinical value, since it could slow or reverse the progression of diseases, such as cancer, which are driven by abnormal cell signaling along a variety of signaling pathways.

“Our study shows the feasibility of targeting a hub in the cell signaling network to reset aberrant cell signaling from multiple pathways and receptors,” said senior author Pradipta Ghosh, MD, an associate professor of medicine.

The UC San Diego team engineered two small protein fragments, known as peptides, to either turn on or turn off the activity of a family of proteins called G proteins. G protein-coupled receptors, which are embedded into the surfaces of cells, are used by cells to sense and respond to their environments. Approximately 30 percent of all prescription drugs target cells by binding to and affecting G protein-coupled receptors.

G protein coupled receptor cycle

Several laboratories, including those at UC San Diego, have discovered that G proteins can also be activated inside cells, and not simply on cell surfaces. Other receptors can activate the internal components of the G protein-coupled receptor, as can a protein called GIV. GIV has been implicated in cancer metastasis and other disease states. Both the “on” and “off” peptides were made from parts of the GIV protein receptor.

In a series of cell culture experiments, the “on” peptides were shown to accelerate the ability of the cells to migrate after scratch-wounding, which is a process linked to wound healing. The “off” peptide, in contrast, reduced the aggressiveness of cancer cells and decreased the production of collagen by cells associated with liver fibrosis. In experiments with mice, the topical application of the “on” peptides helped skin wounds heal faster.

“The takeaway is that we can begin to tap an emerging new paradigm of G protein signaling,” Ghosh said.