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Cardiovascular Diseases: Recent Developments in Regenerative Medicine

Anjum Mahmood,1 Hiteshree Pandya,1 Rajasekar Seetharaman,1Divyang Patel,1 Anand S Srivastava2
1GIOSTAR Research Pvt Ltd., India
2Global Institute of Stem Cell Therapy and Research, USA
Received: July 03, 2017 | Published: July 31, 2017

Correspondence: Anand S Srivastava, Global Institute of Stem Cell Therapy and Research, 4370 La Jolla Village Drive San, Diego, CA 92122, USA, Email

Citation: Mahmood A, Pandya H, Seetharaman R, et al. Cardiovascular diseases: recent developments in regenerative medicine. J Stem Cell Res Ther. 2017;3(2):241‒244. DOI: 10.15406/jsrt.2017.03.00095

Abstract

Advent of regenerative medicine has opened new therapeutic interventions for patients with cardiovascular diseases. The stem cell based cardiac repairing and regeneration along with improved vascularisation can improve diseased heart. Clinical studies over last decade have evaluated the cardiogenic potential of embryonic, induced pluripotent, cardiac, mesenchymal and bone marrow derived stem cells. The clinical studies have raised the hope of successful translation of regenerative cells from bench to bed side. Intrinsic factors guiding the stem cells to site of injury i.e. homing, transformation into cardiomyocytes, and angiogenesis have been assessed in recent years. However, complete understanding of mechanisms co-relating and regulating the process need to be explored. This review focuses on characteristics of stem cells under investigations, for clinical trials, analyzing safety, feasibility, efficacy and mechanism underlying cardio protective and cardio regenerative process. Further, the approaches involving scaffold based 3-dimensional cardiac generation will also be analysed.

Keywords: mesenchymal stem cells, cardiomyocytes, cardiac regeneration, embryonic stem cells, pluripotent stem cells, isscr

Abbreviations

CSCs, cardiac stem cells; BMMNCs, bone marrow mononuclear cells; ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; MSCs, mesenchymal stem cells; bFGF, basic fibroblast growth factor; IL, interleukin; TNF, tumor necrosis factor; ISSCR, international society for stem cell research

The heart failure is one of leading cause of morbidity and mortality all over the world. More than 5million patients are suffering from chronic heart failure post myocardial infarction caused due to ischemic heart disease.1 The disease develops over a period of time due to loss of cardiomyocytes. The condition is further aggravated due to complications related to obesity, hypertension, diabetes, smoking and alcohol consumption. The currently available treatments regime include application of β-blockers, targeting of rennin-angiotension-aldosterone system using ACE (angiotensin converting enzymes) inhibitors, ARBs (angiotensin II receptor blockers) and aldosterone antagonists.2 In selected patients cardiac resynchronization therapy and implantable defibrillators are also recommended.3 Though, the treatment improves condition of patients symptomatically, no remarkable change in mortality or morbidity is observed. Heart transplant is not feasible option due to unavailability of donors and possible immune rejection. New interventions, based on stem cell driven regeneration appear to be promising in current scenario. The cardiac regeneration can bring endogenous repair through formation of new cardiomyocytes and improved vascularisation.

The regenerative capacity of adult human myocardium was considered to be limited as myocardial cells are terminally differentiated. However, discovery of cardiac progenitor cells developed the possibility of heart tissue repair through cardiomyocytes generation. The process involves replacement of damaged myocardial cells with new one, derived from pluripotent cells. The major source for pluripotent cells are human embryonic (hESCs) and induced pluripotent stem cells (iPSC). The hESCs are considered to be promising candidate for cardiac cells development as their cardiogenic potential is established and further they can differentiate into multilineage smooth muscle and endothelial cells, required in myocardial tissue However, clinical use of hESCs and iPSCs still needs detail investigation to assess safety and efficacy. hESCs are derived from embryo which differs from parent in their genome. Similarly iPSCs also demonstrate genetic variability. The risk of immune rejection in such genetically variable cells is high. Both these cells type, if infused in undifferentiated state can lead to teratoma formation.4,5 Another aspect which limits the application of hESCs is ethical issues associated with use of embryonic cells.

Bone marrow derived stem cells

Bone marrow is potential source of stem cells comprising of mixed population of cells mainly primary early stage committed cells, hematopoetic stem cells, endothelial progenitor cells and MSCs. Several studies have evaluated the functional myocardial activity by infusing bone marrow mononuclear cells (BMMNCs). In one major clinical trial, REPAIR, patients with acute myocardial infarction (AMI) were treated with bone marrow derived stem progenitor cells. 204 patients received intracoronary administration of autologus stem cells. After one year, significant reduction in occurrence of adverse events and improved vascular repair was observed. These beneficial effects were observed even after two years of follow up.6–9 Similar, results were observed in different studies using administration of CD34cells in patients of angina and myocardial ischemia demonstrated improvement in frequency of angina and exercise tolerance.10,11 However, despite of moderate success in bone marrow derived stem cells, some of the trials demonstrated little or no improvement after cell transplant. In a set of randomized trials, under category FOCUS-CCTRN and Late TIME trials, BMMNCs were infused to assess several parameters including myocardial perfusions, oxygen consumption and left ventricular function. The end result of trials concluded that cellular infusion did not improve oxygen consumption and left ventricular dysfunction.12–14

Mesenchymal stem cells

Adult cells like mesenchymal stem cells (MSCs) are also capable of driving heart repair. MSCs are characterized by self renewal ability, low immunogenicity, no transplant rejection in host body and low tumorigenicity. They lack major histo-compatibility complex II (MHC II) and B7 co-stimulatory molecule expression, so can easily escape the immune system and overcome host rejection. Though, MSCs lack the ability to differentiate into cardiomyocytes, they contribute to neo vascularization and cardiomyocyte protection.15 Several studies have investigated the ability of MSCs in improving cardiac functions. In PRECISE trial, patients with ischemic cardiomyopathy were treated with adipose derived regenerative cells. Maximum oxygen consumption, ventricular function and exercise capacity was improved in treated group patients which suggested a reduction in inducible ischemia up to 18months.16 Some studies used allogenic source of MSCs as they are considered to be immunoprivileged and immunosuppressed.17,18 A comparative study analysed two types of bone marrow transplants using allogenic and autologus source of cells. In this randomized phase 1/2 POSEIDON study, both types of cells demonstrated safety and potential to regenerate by reducing infarct size and ventricular remodelling.19 In another study, Cardiopioetic stem Cell therapy in heart failure (C-CURE), bone marrow derived MSCs, treated with a particular cardiogenic cocktail, were used for infusion in patients with heart failure. The safety and feasibility was observed, with indications of benefit. Though some concerns were raised about the methodology of trial, the reply by investigators was submitted and published in same journal. Meanwhile, Cardio3biosciences, which conducted C-CURE trial, is working on phase III study of trial.20,21

Cardiac stem cells

Another group of stem cells are categorized as cardiac stem cells (CSCs). They are heterogeneous group of cells, residing in specific heart areas as atria or pericardium and are characterized by expression of c-kit surface marker.22,23 Several trials used CSCs in their clinical study. Stem Cell Infusion in Patients with Ischemic Cardiomyopathy (SCIPIO) trial used c-kit+ CSCs in patients with heart failure due to IHD in phase 1 trial. The results were encouraging as it suggested intracoronary infusion of CSCs led to improved LV systolic function and reduced infarct size. An unprecedented increase in viable myocardium was reported due to therapeutic regeneration.24 At almost same time, Makkar et. al. published a report demonstrating the use of cardiosphere derived cells (CDCs) in patients with left ventricular dysfunction in a randomized phase 1 trial of cardiosphere derived cells for heart regeneration after myocardial infarction (CADUCEUS). The results demonstrated that infusion was safe and an increase in viable myocardial tissue was observed. The scar size reduction was claimed to be 3-5 times better than in case of bone marrow mononuclear cells used in other studies.25

The clinical assessment of stem cell based intervention demonstrated feasibility and safety of transplant; however, the efficacy remained an issue. The variable efficacy of transplants can be understood on basis of mechanism governing mode of action of administered cells.

Mechanism underlying myocardial activity conferred by stem cell transplants

Cardiogenic cells including ESCs and iPSCs stimulate heart repair by stimulating differentiation to cardiomyocytes. However, exact mechanism regulating the process is not completely understood. Paracrine signalling has been attributed main driving force behind cardiac repair. The mechanism can be categorized into cardio protection, angiogenesis, myogenesis and endogenous repair. The cardio protection is conferred by secretory anti-apoptotic factors. Once inside body, MSCs inhibit activation of transcription factor NF-KB in B cells, which in turn attenuates secretion of pro-inflammatory, factors TNF-α and IL-6, and promotes anti-inflammatory cytokine IL-10. TNF-α and IL-6 are toxic to cardiomyocyte activity and cause reduced contractile activity and induce apoptosis.26MSCs regulate immune response and induce IL-10 secretion through monocytes and macrophages which in turn inhibits NF-KB nuclear factor. Along with cardioprotection, MSCs secrete angiogenic vascular endothelial growth factor (VEGF) and arteriogenic basic fibroblast growth factor (bFGF). VEGF increases capillary wall permeability, induces cell proliferation, migration and vascularisation. On other hand, bFGF promotes smooth muscle formation as part of angiogenesis.27,28

Myocardial infarction leads to accumulation of collagen resulting into fibrosis. Under fibrotic environment expression of many genes, growth factors and cytokine is downregulated resulting into inhibition of endogenous cardiac repair.29 Under such environment, MSC’s transplantation brings about modulation of matrix by metalloproteinase resulting into matrix degradation activity. MSCs control fibroblast activity by down regulating synthesis of type I and type III collagen synthesis and finally inhibit ventricular remodelling. Along with matrix regulation, MSCs cause endogenous cardiac repair through stimulation of, c-kit+ cardiac stem cells and cardiomyocyte cell division.30,31

Researchers have proposed another way through which MSCs may impart cardiac repairing function. The MSCs along with other factors, secrete exosomes which are vesicles containing biological molecules primarily proteins, mRNA, microRNA and lipid.32Exosomes play vital role in cell-cell interaction and mediating bidirectional exchange of material. These exosomes induce angiogenesis in endothelial cells by direct transfer of mRNA. Timmers L et al.33 demonstrated that media conditioned with hMSC reduce infarct size by 59% in animal model of myocardial ischemia.33

The safety, feasibility and efficacy of stem transplant in cardiovascular diseases have been studied in numerous trials using wide range of stem cells. However, each approach is associated with various benefits and limitations. Though, ESCs and iPSCs are potentially capable of generating new cardiomyocytes, infusion of undifferentiated cell may lead to teratomas formation. Further, use of ESCs involves ethical concerns. Adult stem cells like CSCs and MSCs appear to be potentially more feasible for clinical transplants. CSCs, directly or indirectly through MSCs can be induced for endogenous cardiac repair. Additionally, they are not associated with tumor formation, which is major advantage. MSCs exerts cardio-protective and regenerative effect through several mechanisms including secretion of growth factors VEGF, bFGF, metalloproteinases, exosomes and stimulation of endogenous c-kitCSCs. Though, MSCs themselves lack the ability to differentiate into cardiomyocytes, they provide beneficiary effect through paracrine signals.

Tissue engineering

Along with cell based approaches for damaged cardiac repair, researchers have explored the concept of in-vitro 3 dimensional generation of complete heart. Emerging trends in tissue engineering indicate towards possible generation of cardiac structures. The methodology involves re-population of cardiomyocytes and endothelial cells around a natural (extra-cellular) or synthetic matrix based scaffold. Reprogrammed iPSCs are primary source of cells, incorporated in engineered construct. The selection of scaffold depends on elasticity, biocompatibility and biodegrability of material. Further, the structure must be interconnected, appropriately porous and supportive for cell proliferation and differentiation of stem cells. The extracellular matrix protein (ECMP) based biomaterials are natural option for scaffolds developments in cardiac engineering and regeneration as they are bio-compatible. However, the decellularized matrices are superior to ECMP based, as later one does not mimic the complexity and structure of native tissue. The decellularized matrixes are obtained through detergent treatment of intact cardiac tissue. The process retains intact vasculature and complex 3D arrangement of collagens, elastin and glycosaminoglycan.34 The decellularized scaffold was successfully tested initially in rat, by recellularization of neonatal cardiomyocytes.35However, human organ generation using decellularized scaffolds is limited due to unavailability of intact hearts. Due to this animal based heart structures closely resembling to humans are under investigation. Synthetic polymers which have been investigated till now are based on poly (lactic acid) (PLA), poly(ethylene glycol) (PEG), poly(caprolactone) (PCL), poly(l-lactide-co-caprolactone) (PLCL), poly(glycerol sebacate) (PGS), and polyurethane (PU).36,37

Future directions

Clinical trials conducted over last few years have generated substantial data on implications of regenerative medicine on cardiovascular disease. Most of data are based on results of early phase I and phase II studies. Contradictory results in several studies have raised concern over methods adopted during trial design. To deal with such issues and bring transparency in clinical trials procedures, International Society for Stem Cell Research (ISSCR) has issued revised guidelines to be followed. These guidelines are aimed to encourage best practices in translational and clinical research. Under current scenario, in many cases, where conventional treatment fails to provide prolonged relief to cardiovascular patients, new interventions are required which can provide less invasive, sustained and cost effective benefits. Stem cell based repair or transplant of in-vitro developed cardiac structure in cardiovascular diseases open a wide array of opportunities to healthcare system where scientists, clinicians and developers can contribute keeping ethical values in practice.

Acknowledgements

None.

Conflict of interest

The author declares no conflict of interest.

References

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Mannose Supplements Induce Embryonic Lethality And Blindness in Phosphomannose Isomerase Hypomorphic Mice

Associated Data

Supplementary Materials

Abstract

Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (∼15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1–2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ∼50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.—Sharma, V., Nayak, J., DeRossi, C., Charbono, A., Ichikawa, M., Ng, B. G., Grajales-Esquivel, E., Srivastava, A., Wang, L., He, P., Scott, D. A., Russell, J., Contreras, E., Guess, C. M., Krajewski, S., Del Rio-Tsonis, K., Freeze, H. H. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice.

Keywords: congenital disorder of glycosylation, MPI-CDG, lens, eye defects

A potentially lethal form of rare congenital disorder of glycosylation (CDG), type Ib [CDG-Ib; or phosphomannose isomerase (MPI)-CDG] can be treated with mannose as a dietary supplement (1). Supplementation overcomes impaired glycosylation caused by hypomorphic mutations in MPI because mannose bypasses the impaired conversion of fructose-6-phosphate (Fru-6-P) to mannose-6-phosphate (Man-6-P), which is the major source of Man-6-P derived from glucose. Mannose alleviates patients’ stunted growth, hypoglycemia, liver dysfunction, coagulopathy, and protein-losing enteropathy (2). Exogenous mannose is converted to Man-6-P by hexokinase (HK), replenishing this deficient precursor needed for multiple glycosylation pathways, including the N-glycosylation pathway, viaphosphomannomutase (PMM2); excess Man-6-P is catabolized by the residual MPI activity (Scheme 1). Patients on this therapy survive and lead a normal life without obvious side effects (2).

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Mannose metabolic pathway. Man, mannose; Glc, glucose; HK, hexokinase; MPI, phosphomannose isomerse; PMM2, phosphomannomutase2; GDP-Man, GDP-mannose; Dol-P-Man, dolichol phosphate mannose; LLO, lipid linked oligosaccharide.

To model MPI-CDG and follow the effects of mannose therapy, we previously knocked out the single Mpigene in mice, leading to death at embryonic day 11.5 (E11.5) due to abnormalities in both placenta and the embryo. Mannose could not rescue because Man-6-P accumulates to toxic levels, limiting ATP and inhibiting several glycolytic enzymes (3). However, because patients with MPI-CDG have residual enzymatic activity, hypomorphic mice would offer a more patient-relevant model than would a complete Mpi knockout (KO). Here, we describe a viable, hypomorphic mouse line containing a patient-derived mutation that reduced enzymatic activity and altered mannose metabolism, as predicted. While a minority of mutant embryos died in utero, surprisingly, adolescent and adult mice had none of the expected symptoms reported in patients with MPI-CDG; these Mpi-deficient mice had a subclinical phenotype. However, if dams consumed mannose during pregnancy, most of the Mpi hypomorphic embryos died, and nearly half of the survivors were born with severe ocular defects. The combination of reduced enzymatic activity and the increased mannose load altered its metabolic flux, leading to Man-6-P accumulation in the eyes.

Mannose is widely used as a “natural” treatment for urinary tract infections; this seemingly innocuous sugar may have a negative effect for some pregnant women. While the frequency of MPI-CDG is unknown, women at risk for having subsequent MPI-CDG children who intend to take mannose as a “prenatal therapy” may inadvertently cause other side effects.

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 Diabetes: Stem Cells Offering Healthy Promises

 

Aditi Saraswat,1 Anand Srivastava2
1Henry Ford Medical Center, USA
2Global Institute of Stem Cell Therapy and Research, USA
Received: April 30, 2018 | Published: May 09, 2018

Correspondence: Anand Srivastava, Global Institute of Stem Cell Therapy and Research, 4660 La Jolla Village Drive, San Diego, CA, 92122, USA, Tel 8583 4424 92, Email 

Citation: Saraswat A, Srivastava A. Diabetes: stem cells offering healthy promises. J Stem Cell Res Ther. 2018;4(2):45‒46. DOI:10.15406/jsrt.2018.04.00113

 

Editorial

Diabetes is a chronic lifelong disease and according to Diabetes Association of America, in 2015 itself approximately 30.3million Americans (9.4% of the population) have the disease. Unfortunately, almost one fourth (or approximately 7.2million) are unaware that they have it. An additional 84.1million people have pre-diabetes. With increasing prosperity, its prevalence has increased in almost all populations of the world and ranges from 5-15%. As it affects so big portion of the world population a long-lasting cure is urgently warranted. People with diabetes need to manage their disease in order to avoid related complications and maintain healthy social and economic interactions.

Diabetes affects individuals of all age groups and has been classified in two types. Type 1 diabetes (T1D) is an autoimmune disease that occurs when a person’s pancreas stops producing insulin. It is usually diagnosed in children and young adults, previously known as juvenile diabetes. Only 5% of people with diabetes have this form of the disease. On the other hand, type 2 diabetes (T2D) is the most common form of diabetes. In patients of T2D, the body does not use insulin properly mostly because of insulin resistance. Because of that, at first, pancreas compensates by making extra insulin. However, over time it isn’t able to keep up and can’t make enough insulin to keep your blood glucose at normal levels.

Diabetes affects every part of the body and causes complications related to heart, brain, kidney, circulatory system etc. Managing diabetes exerts a significant burden on the economy in general. During 2017, according to an estimate, diabetes-related care of people directly or indirectly could have costed around $327 billion.1 Though a number of medications are already in clinical use but none of them grant a long-term cure and all of them have some or other undesired side-effects.

 Since almost all pharmacological drugs, irrespective of the target molecule in the pathway involved in the manifestation of diabetes-related complications, have some side effects a safer and comparatively long last therapeutic alternative is desperately needed. To meet the continuous need for insulin, pancreatic transplants have been tried which turned out to be very cost-intensive and impractical because the donor pancreases have to be recovered from suitable cadavers and then transplanted.2 Logically, transplantation of a tissue from other individual comes up with so many concerns like compatibility of a graft in the new host and its survival as immune rejection is usually a valid concern most of the time. To circumvent all these concerns another alternative way of handling the problem was needed for a long time. Discovery of stem cells and related extensive research has offered a ray of hope to manage the problem efficiently with a sound possibility of a permanent cure.

Stem cells, depending on the source of their origin, are classified as embryonic or adult or induced. Embryonic stem cells are capable of differentiating in all cell types for a body while adult cells which have attained some tissue-specific differentiation, lose that ability slightly. Since a number of ethical issues crop up with use of embryonic cells, adult stem cells are next best type of cells to lean back on. Another favorable factor for using adult stem cells is that these can be isolated from tissues which are easy to extract from an individual like belly fat or bone marrow. Cells of these origins are classified as mesenchymal stem cells (MSCs). MSCs are known to promote the regeneration of pancreatic islet beta cells, protect endogenous pancreatic islet beta cells from apoptosis, and ameliorate insulin resistance of peripheral tissues by providing a supportive niche microenvironment driven by the secretion of paracrine factors or the deposition of extracellular matrix.3,4

In general, implantation of MSCs can alleviate T2D by a number of mechanisms. These cells, if implanted directly in the pancreas, thanks to their multipotential ability to differentiate in diverse types of cells of their immediate vicinity, can produce new insulin-producing cells. Investigators, in order to promote the chance of differentiation of cells in insulin-producing cells, have preprogrammed MSCs by culturing in serum-free high glucose media or neuron conditioned media before transplantation. Intravenous infusion of stem cell has been shown to regenerate beta cells of islets in rats5 also promote the survival during hypoxia and oxidant stress.6 In addition to these effects, infusion of stem cells has been shown to promote insulin sensitivity.7 Though the exact mechanism by which stem cell bring about increased insulin sensitivity is not deciphered, it could be because of stem-cell-mediated decrease in systemic inflammation as it is well established that insulin resistance is strongly correlated with chronic low-grade inflammation.

Encouraging findings in cases of diabetes treatments with stem cell therapies have led the clinicians to try implantation or infusion or both in the clinical set up also. On the clinical trial site of NIH, more than 150 trials at different stage have been listed. MSCs of diverse origins either were implanted directly in the pancreas8 or were infused in blood stream9 or both10 showed promising results up to 12 months of follow up. A couple of clinical parameters are often used to ascertain the effectiveness of a therapy in cases of diabetes. A decrease in Hb A1C is one of those parameters which were used by Estrada et al.11 and they reported a significant decrease.11 In another study, insulin need decreased or was abolished altogether.8 Similarly, implantation or infusion of MSCs has been shown to improve pancreatic function i.e. increased insulin production. Same time, increased insulin sensitivity is also attained by MSCs.

Just like other medical helps, stem cell therapy can have some undesirable effects, though the incidences are few and far between. Even those undesired effects, which happen after stem cell transplantation are very mild and easily manageable like mild to moderate fever or nausea or headache.

In conclusion, stem cell therapy does offer a long lasting therapeutic alternative for treating T2D. Same time it has to be kept in minds of both clinicians and patients that it is not a permanent cure. T2D is a metabolic syndrome which manifests after a long duration of unhealthy life style which needs to be addressed in order to lead a healthy life. Compared to all other available therapies, stem cell therapy can offer a lot longer period for individuals to develop a healthy life style which would help fend off re-occurrence of the disease.

Conflict of interest

 

Author declares that there is no conflict of interest.

References

 

  1. Enocrinology Advisor. Total Estimated Cost of Diagnosed Diabetes $327 Billion in 2017. Endocrinology Advisor. 2017.
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Role of Cell Based Approaches in Cancer Immunotherapy

Anjum Mahmood,1 Anjani Srivastava,2 Shivangi Srivastava,2Hiteshree Pandya,1 Neel Khokhani,1 Divyang Patel,1 Rangnath Mishra3
1GIOSTAR Research Pvt Ltd, India
2Global Institute of Stem Cell Therapy and Research, USA
3Department of Medicine, National Jewish Health, USA
Received: February 17, 2017 | Published: May 05, 2017

Correspondence: Rangnath Mishra, Department of Medicine, National Jewish Health, Denver, CO 80206, USA, Email

Citation: Mahmood A, Srivastava A, Srivastava S, et al. Role of cell based approaches in cancer immunotherapy. J Stem Cell Res Ther. 2017;2(5):145‒147. DOI: 10.15406/jsrt.2017.02.00077

Abstrat

Immunotherapies hold the potential for cancer treatment since their mode of action is distinct to chemo and radiation therapy and largely depends on harnessing body’s own immune system. The major advantage associated with cancer immunotherapy is that cell responses are specific to tumor and with low or negligible toxicity. Preclinical and clinical studies have evidenced that modulation of immune system can subvert the immunosuppressive environment under progressive tumor conditions. The modulation can be brought into several ways including infusion of ex-vivo or in-vivo activated antigen presenting cells (dendritic cells), immune checkpoint antibodies, adoptive transfer of T cells, genetically modified T cells, cancer cell vaccines, stem cells, cytokines and others. In this review, we will keep the discussion focused to some of cell based approaches.

Keywords: immunotherapy, dendritic cells, adoptive t cell therapy, mesenchymal stem cells, cancer

Abbreviations

DCs, dendritic cells; MSCs, mesenchymal stem cells; MDSCs, myeloid derived suppressor cells; BBB, blood brain barrier; CAR, chimeric antigen receptor, IFN, interferon; TILs, tumor infiltrating lymphocytes; CSCs, cancer stem cells; PBMCs, peripheral blood mononuclear cells; IL, interleukin

Introduction

An integrated immune system prevents development and progression of neoplastic cells in a process termed as immune surveillance. T-cells play an important role in detecting and eliminating tumor cells. In turn, they are dependent on dendritic cells for tumor antigen presentation and activation signals to stimulate them. One of the most important reasons behind failure of cancer immuno-surveillance is hampered T-cell activity due to lack of co-stimulatory activation signals by dendritic cells resulting into peripheral tolerance. Other factors driving tumor progression include immunosuppressive tumor micro-environment, infiltration of regulatory T cells, release of immunosuppressive cytokines like IL-10 and TGF-β, reduced expression of MHC molecules, myeloid derived suppressor cells (MDSCs) and heterogeneity of tumor sub-clones at the genetic level. Studies have shown that expansion of Treg cells is associated with poor prognosis and reduced survival. Similarly, abnormal accumulation of MDSCs is also correlated with tumor evasion mechanism. Though, chemotherapy is first line of treatment, the efficacy is restricted later due to development of drug resistance. The major reasons for resistance development includes drug-targeted gene amplification (e.g. BRAF gene) and substitution mutation in some cancer cells leading to the escape of drug cytotoxic effect.1Further, non-specific cytotoxicity of chemo agents result into lymphodepliton. To address all these issues, new therapeutic interventions are required which alone or in combination alter the tumor microenvironment to enhance beneficial effects without causing toxicity. In this context, immunotherapy is expected to play significant role. Cancer immunotherapy can be defined as set of techniques aimed to eliminate malignant tumors through mechanisms involving immune system responses. The agents driving immune alteration are termed as immunomodulators. In this review, we will discuss briefly some of specific methods mediating immunomodulation including dendritic cell based approaches, adoptive T cells transfer and mesenchymal stem cells based targeted delivery of drugs.

Dendritic cells

The dendritic cells (DCs) based immunotherapeutic approach has emerged as one of alternative treatment options owing to its low toxicity in comparison to other standard methods. The clinical efficacy has been demonstrated with improvement in overall survival rate and low toxicity. The application of ex-vivo-generated DCs emerged in an effort to improve the therapeutic efficacy in cancer patients in whom the dysfunction of endogenous DCs is commonly observed. The DCs are generated using several approaches. The most common used method is ex-vivo differentiation of DCs from peripheral blood mononuclear cells (PBMCs) using interleukin-4 (IL-4) and granulocyte macrophage colony stimulating factor (GM-CSF).2 Other ways of generating ex-vivo DCs is to derive it from progenitor CD34+ cells or in-vivo stimulation using C-type lectin receptors (CLRs) present on DC surfaces. CLRs specific antibodies attached to tumor antigens are readily internalized by DCs and generate antigen specific antitumor immunity.3,4

In terms of clinical success, initially most of phase I/II studies demonstrated only safety and feasibility. The efficacy remained an issue in phase III due to inconsistent and inconclusive data. The first successful commercialized product was Sipuleucel-T for castration resistant prostate cancer. The vaccine was approved by FDA in 2010.5 The approval was based on phase III results of IMPACT (Immunotherapy Prostate Adenocarcinoma Treatment) trials. Later, several other studies demonstrated beneficial effect of DC immunotherapy in head and neck squaous cell carcinoma,6 uterus,7 prostate8and breast cancer and Her-2 positive ductal carcinoma in-situ.9 However, for most cancers, preclinical success could not be translated up to phase III due inconstant data and less optimized process.

Adoptive t cell therapy

Adoptive transfer of T cells is a potent treatment option for metastatic tumors. The T cell based interventions are specific, robust (undergoing upto 1000 fold clonal expansion) and retain memory. Further, T cells can infiltrate to the site of antigen and thus holds capacity to eradicate distant metastasis. In chimeric antigen receptor (CAR) approach T cells are engineered cells which provide specificity to the effector cells. Most of clinical investigations targeted B cell related malignancies using CD19 directed CART cells. These studies demonstrated response in many patients.10–12 Antigens like human epidermal growth factor receptor over-expressed in tumors like breast, ovarian, non small cell lung carcinoma (NSCLC), salivary gland, pancreatic and endometrial cancers are under investigation for CART cell development.13–17 Till now, most of the success of CART cells is limited around hematological malignancies, a huge scope is still available for exploring new antigens, directed to eliminate metastatic, resistant and non-hematological malignancies.

Tumor infiltrating lymphocytes (TILs) are found in the tumor region and are associated with anti-tumor activity. They are isolated from tumor, expanded under ex-vivo conditions, screened for anti-neoplastic activity and infused back into patients. Their presence in tumor is associated with better clinical outcome. These lymphocytes at tumor site recognize the antigens presented by MHCI and MHC II molecules on cell surfaces. TILs raised against melanoma recognize antigens especially MART-1, gp100 and tyrosinase.18–21 Another class of antigens termed as cancer/testis (C/T) antigens are also recognized by melanoma TILs. The class includes several antigens like MAGE, NYESO-1, RAGE, SAGE and SSX2.22,23 Rosenberg et al was pioneer in isolation and expansion of melanoma specific TILs developed for clinical purposes.24 Rosenberg et al.,24 conducted three sequential clinical trials, in which 93 patients (metastatic melanoma) were treated with lympho depleting preparative regimen, autologous TILs and IL2. Objective response rates by RECIST criteria in the three trials were 49%, 52% and 72%, respectively. Study showed that 22% of all patients achieved complete tumor regression and 19% of the patients were disease-free for more than three years.25 Till now, most of the clinical investigations focused on melanoma due to considerable success. However, non melanoma tumors demonstrated less feasibility due to lack of reproducibility of TIL generation from primary and metastatic tumors.

Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are adult stem cells with unique characteristic ability of homing, facilitating their application in cancer immunotherapy. These adult stem cells are reported to migrate at site of inflammation, injury, infection and tumors where they immunomodulate the immediate micro-environment through secretion of soluble factors.26 The therapeutic value to MSCs is conferred by transportation of anti-tumor genes. MSCs act as delivery vehicle for many tumor inhibiting genes and factors to tumor site.27 They offer therapeutic advantage of ease of isolation, ex-vivo expansion, transduction and transplantation. The movement of MSCs to tumor site is driven by chemotactic factors, chemokines and chemo-attractants released by progressive tumors.28 MSCs hold another characteristic feature which makes them a favorable tool for carrying targeted anti-cancer gene i.e., they are immunoprivileged. The absence or low expression of MHC II, MHC I, CD80, CD40, and CD86 molecules on cell surface make them undetectable by host immune system. Further, immunoprivileged nature also confers possible use of allogenic MSCs. However, at same time, MSCs are immunosuppressive, which exerts significant effect on host disease. In case of graft versus host disease, transplant of MSCs offer a promising treatment, where disease can develop due to histo-compatibility mismatch.29 On other hand, application of MSCs can induce tumor progression due to immune inhibition.

Several genes demonstrating therapeutic efficacy in preclinical models have been tested for expression in MSCs as vehicle. The genes which have been engineered in MSCs to target tumor sites include IL-12, VEGFR-1, CX3CL1, HSV-Tk, TRAIL and IFNβ.30–33 Their expressions were related to localized and metastatic tumor inhibition and survival benefits in tumor models. HSV-Tk (herpes simplex virus thymidine kinase) is a pro drug converting enzyme which is delivered through MSCs along with systemic administration of ganciclovir. In this approach, which has been successfully tested in glioma and pancreatic cancer, MSCs carry the suicide enzymes to the tumor site thus avoiding systemic toxicity.34 In brain tumors like glioma where blood brain barrier (BBB) restrict passage of anti tumor therapy, MSCs based delivery of drugs can provide therapeutic solutions.35

Future directions

Recent advances in understanding the mechanism underlying tumor progression and role of immune system has laid the foundation of immunotherapy based interventions in clinical malignancies. By adopting unique immunotherapeutic approach specific to diseased condition and optimal conditions of delivery significant level of benefits can be expected. Further, exploration of new targeted strategies is also required to extend scope of application and avoid unwanted adverse events in patients. The targeting of other identified DC cell surface receptors like mannose receptor (MR), CIRE, DC-SIGN, DCIR, LSECtin, L-SIGN, Langerin, Dectin, DNGR-1, MICL, MGL CLEC2, CLEC12B, LOX-1, BDCA-2, DEC205, scavenger receptor, DC-ASGPR, FIRE, DC-STAMP and Toll-like receptors (TLRs) will definitely open the new dimensions in in-vivo DC based approaches.5 Further, targeting of cancer stem cells (CSCs) via DCs will also improve specificity of anti-tumor activity. Similarly, role of MSC derived exosomes in delivery of therapeutic agents is also currently under investigation in several studies. Exosome-mediated delivery of tumor suppressor miRNAs and targeting of growth-regulatory pathways, such as the Wnt and Hedgehog pathways, as well as angiogenic pathways, such as the VEGF and kinase pathways, could be novel strategies to monitor tumor growth. In light of current knowledge and advances in cancer immunotherapy we conclude that under optimal conditions, tangible benefits can be realized in cancer management.

Acknowledgements

None.

Conflict of interest

The author declares no conflict of interest.

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Mesenchymal Stem Cell Conditioned Media Ameliorate Psoriasis Vulgaris: A Case Study

 

Seetharaman R, Mahmood A, Kshatriya P, Patel D, Srivastava A. Case Reports in Dermatological Medicine. May 2019; Article ID 8309103. https://doi.org/ 10.1155/ 2019/ 8309103.

Case Reports in Dermatological Medicine

Volume 2019, Article ID 8309103, 5 pages
https://doi.org/10.1155/2019/8309103
Case Report

Mesenchymal Stem Cell Conditioned Media Ameliorate Psoriasis Vulgaris: A Case Study

1GIOSTAR Research Inc. Pvt. Ltd., Ahmedabad, Gujrat, India
2Global Institute of Stem Cell Therapy and Research, 4660 La Jolla Village Drive, San Diego, CA 92122, USA

Correspondence should be addressed to Anand Srivastavaanand@giostar.com

Received 6 April 2019; Accepted 21 April 2019; Published 2 May 2019

Academic Editor: Jacek Cezary Szepietowski

Copyright © 2019 Rajasekar Seetharaman et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Psoriasis, an autoimmune disease, affects a vast number of peoples around the world. In this report, we discuss our findings about a scalp psoriasis suffering patient with a Psoriasis Scalp Severity Index (PSSI) score of 28, who was treated with Mesenchymal stem cell conditioned media (MSC-CM). Remarkably, complete regression was recorded within a treatment period of one month only (PSSI score of 0). A number of bioactive factors like cytokines and growth factors secreted by MSCs in the conditioned medium are very likely to be the principle molecules which play a vital role in skin regeneration. Treatment using MSC-CM appears to be an effective tool for tackling the psoriatic problem and, thus, may offer a new avenue of therapy which could be considered as an alternative approach to overcome the limitations of the cell-based therapy.

1. Introduction

Psoriasis is a chronic disease thought to be of autoimmune origin which is characterized by patches on the skin and nails. It has been considered as a serious skin related problem affecting approximately 100 million individuals worldwide. About 2% of the world population and 0.44-2.8% of the Indian population were affected by psoriasis in 2016-2017 [12]. Plaque, guttate, inverse, pustular, and erythrodermic are the five major types of psoriasis. Plaque psoriasis, also known as psoriasis vulgaris, is the most common form of the disease (about 90% of the cases) [3] which typically presents with red patches with white scales on top. Psoriasis vulgaris which commonly affects the areas includes scalp, knees, elbows, hands, nails, and feet [4].

Psoriasis, an autoimmune-inflammatory disease probably predisposed due to genetic makeup, is mediated by T-helper cells. Polymorphism, referred to as differences in DNA sequences of a gene, can be incurred by various external agents like chemicals, viruses, or radiation. Polymorphisms in genes of Th2 cytokine/regulatory T-cell (interleukin-10/IL10), Th1/Th17 cytokine (IL-12B and IL-23R), and tumour necrosis factor alpha (TNFAIP3; TNIP1) confer which increased other risks like cardiovascular diseases amongst psoriasis patients [57]. Single nucleotide alteration caused polymorphism in Th1 proinflammatory cytokine gene IL-2 [–330 (G/T)] which has been shown to be associated with greater disease severity in the Indian population [1]. On the other hand, another gene polymorphism occurring in Th-2 cytokine/regulatory T-cell (IL-4) has been shown to be protective against psoriasis [5]. Upregulation in the levels of inflammatory cytokines leads to psoriasis which also can be associated with an increased risk of psoriatic arthritis, lymphomas, cardiovascular risk, Crohn’s disease, and depression [3]. There is no permanent cure for psoriasis, though steroid creams, vitamin D3 cream, ultraviolet light, and immune system suppressing medications (methotrexate) have been in wide use to help control the symptoms with some success [89].

Mesenchymal stem cells (MSCs) are multipotent adult stem cells which have an excellent capacity to proliferate for an extended period of time while maintaining the undifferentiated cell status. The resulting daughter cells can differentiate into various types of cells of host tissues and thus help repair wear and tear incurred [10]. MSCs have a potential to serve as a powerful tool in cell-based therapy due to their tissue regenerative and host immune modulatory capabilities. The functions exhibited by MSCs have attracted a number of scientists and clinicians to investigate the mechanisms involved in their curative and tissue regeneration functions. A very few articles have reported the effectiveness of stromal vascular fraction (SVF)/MSC therapy in curing psoriasis by regulating the immune systems. Lee et al. [11] reported that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) ameliorate psoriasis-like skin inflammation in mice and have regulatory effects on immune cells including CD4+ T cells and dendritic cells. The first case study on intravenous infusion of SVF into psoriasis patient demonstrated a significant decrease in symptoms with a noticeable difference in skin appearance, psoriasis area, and severity index (PASI) score reduction (from 50.4 to 0.3) [12]. Chen et al. [13] reported that umbilical cord-derived MSC (UC-MSC) infusion effectively reduced psoriasis in human subjects. It was believed that MSCs’ migration into the skin lesions and their immunomodulatory, autoimmune inhibitory, and paracrine effects were the principal factors behind the ameliorative effects. Other recent preclinical studies have shown that stem cell-derived conditioned media (CM) exhibit effective healing of psoriasis-like wounds and thus CM is an alternative for several cell-based therapies [1415]. The paracrine factors including growth factors, chemokines, and cytokines secreted from stem cells play a major role in wound healing [16] and these molecules are present in CM or spent medium harvested from cultured cells [17]. In short, CM can serve as a novel treatment approach in regenerative medicine which has been shown to have a successful outcome in preclinical studies. However, a very few reports are available on the clinical application of CM for treatment of any disease. Based on the principles and importance of CM, the present study was aimed at investigating the effect of MSC-CM on a patient suffering from psoriasis. This study is believed to be the first clinical report on the use of MSC-CM to treat psoriasis.

2. Case Report

2.1. Patient

A 38-year-old male patient, who was suffering from psoriasis vulgaris for 2 years, paid a visit to our centre. Preliminary examination of the patient showed that numerous erythematous plaques with numerous silvery scales present all over the scalp including the area behind the ears. The severity of the disease was assessed to be 28 on Psoriasis Scalp Severity Index (PSSI), calculated by the standard method which combines the severity (erythema, induration, and desquamation) and percentage of affected area.

2.2. Preparation of MSC-Conditioned Media

Adipose tissue was collected from a healthy volunteer by lipoaspiration by a plastic surgeon under the aseptic conditions in the O.T. About 100 ml of fat was aspirated out from the waist area and collected in a sterile container. The fat tissue contacting stem cells was processed in a biosafety laminar airflow chamber. MSCs were isolated from adipose tissue by standard enzymatic digestion method with 0.1% collagenase type I. Following the centrifugation, the resulting pellet was cultured in DMEM medium (Invitrogen, Paisley UK) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin, at 37°C in humidified atmosphere containing 5% CO2. The media were changed after every 3 days. About 5×106 MSCs of passage 2 were seeded in each T175 culture flask (n=10) containing 30 ml of DMEM medium supplemented with 10% FBS. MSCs were confirmed with spindle shaped morphology and free from any contamination (Figure 1) using a phase-contrast microscope. When cells attained 90% confluence at passage 2, the culture media were replaced with serum-free DMEM. After 72 h of incubation, resulting MSC-CM was collected, centrifuged at 2000 rpm for 5 min to remove the cell debris, filtered through 0.22-μm filter, and then concentrated (10 times) by ultrafiltration using centrifugal filtering units with a cut-off value of 3 kDa (Amicon Ultra-15; Millipore, MA), according to the manufacturer’s instructions. The concentrated MSC-CM was aliquoted and stored at -20°C until use. MSC-CM was topically applied on the afflicted areas once a day over a period of one month. Clinical parameters like severity, changes, and clearance of psoriatic plaques were monitored at regular intervals.

Figure 1: Phase-contrast microscopic image showing spindle shaped MSCs (× 100).
2.3. MSC-CM Ameliorates Psoriasis Vulgaris

Numerous psoriatic erythematous plaques with adhering silvery scales over the different regions of the scalp were observed before the treatment regimen started. In general, the number of the scales declined significantly within 2 weeks of topical application of MSC-CM. Interestingly, clearance of silvery scales and severity of psoriatic plaques were completely abolished within one month of the treatment (Figure 2). The PSSI score reduced to 0 from 28 and regression of the disease continued for 6 months of follow-up. The patient did not take any other medication during the follow-up period of six months and led an improved quality of life without any adverse side effects.

Figure 2: Effect of MSC-CM on psoriasis vulgaris. The scalp of patient showing numerous erythematous plaques with adherent silvery flakes before MSC-CM-treatment (a, b). Regression of psoriasis and a complete clearance of inflammatory erythematous plaques recorded after topical application of MSC-CM for a period of one month (c, d).

3. Discussion

Psoriasis is an autoimmune disease mediated by hyperactivity of T-helper cells. Increases in the levels of inflammatory cytokines triggered by T cells lead to psoriasis and other associated diseases [3]. Therapies using multipotent MSCs have been shown to be effective in treating psoriasis and psoriasis-like other skin diseases. Clinical benefits may be attributed to MSCs engraftment or to their paracrine/immunomodulatory effects. However, transplanting MSCs come with few limitations like low survivability of cells in the host due to harsh microenvironment and cell loss because of poor or no cell adhesion [18]. Hence there is need of the hour to find an alternative for cell-based therapy.

Cell-free products of MSC origin are effective in wound healing and skin diseases. In this study, we demonstrated that MSC-CM can be used to treat patients with chronic psoriasis. Prior to MSC-CM treatment, the patient had received different medications but without any noticeable effective outcome. Remarkably, topical application of MSC-CM for a period of only one month completely abolished the erythematous plaques and resulted into a complete clearance of adherent silvery scales over the scalp. Further, the severity of psoriasis was completely reduced, from PSSI score of 28 to 0. This regeneration of tissue and improvement of qualitative appearance skin may be mediated by the growth factors, chemokines, and cytokines present in MSC-CM. Previous reports have shown that the paracrine factors secreted by MSC present in CM play a vital role in the healing of psoriasis-like wounds [1617]. Kim et al. [19] stated that adipose-derived stem cell (ADSC)-CM has regenerative effects on skin wounds. It stimulates both collagen synthesis, migration of dermal fibroblasts and promotes wound healing in animal models. ADSC-CM also upregulates the transcription of type I procollagen-alpha-1 chain gene of fibroblasts and induces Rho-associated kinase (RhoA-ROCK) signalling pathway, which leads to the proliferation of keratinocytes and dermal fibroblasts. In other studies [2021], MSC-CM promoted the recovery of skin burn wounds in rats, marked by an acceleration of wound closure, greater numbers of fibroblasts around and injured tissue and blood vessels, high epithelialization ratio, and high density of collagen fibres. It was suggested that basic fibroblast growth factor (bFGF) played an important role in the tissues regeneration of skin burn treated by MSC-CM.

In vitro and in vivo studies involving UC-MSC-CM demonstrated that its application caused an increase in the proliferation and migration of dermal fibroblasts, decrease in the ratio of transforming growth factor-β1/β3, and an increase in the ratio of matrix metalloproteinase over counter agent tissue inhibitor of metalloproteinases [22]. Similarly, human embryonic stem cell (hESC)-derived endothelial precursor cells CM is a rich source of a number of growth factors like epidermal growth factor (EGF), bFGF, fractalkine, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6. It was successfully used in the treatment of excisional wound healing in rats [23]. A comparative study revealed that wound healing by bone-marrow derived mesenchymal stem cell (BMMSC)-CM was significantly higher than that by fibroblast-CM [16]. The fact that BMMSC-CM had higher levels of paracrine factors than those in fibroblast-CM indicated the importance of origin of cells played a significant role in the production of paracrine factors. Other growth factors like Vascular endothelial growth factor (VEGF), insulin like growth factor (IGF), EGF, keratinocyte growth factor (KGF), angioprotein-1 (Ang-1), stromal derived factor-1, and erythropoietin (EPO) were also present in BMMSC-CM. With the supporting evidence of previous reports, the ameliorative effect of MSC-CM exhibited in this present study could be attributed to the presence of numerous growth factors secreted by MSCs in the media.

4. Conclusions

This is the first case report which demonstrates the ameliorative effect of MSC-CM on psoriasis vulgaris. MSC-CM is likely to have a wide range of cytokines and growth factors which can directly act on resident skin cells and thus can help in the skin regeneration. The active bioactive ingredients and their needed combination are yet to be determined. Use of MSC-CM instead of direct implantation of MSCs to tackle the issue offers an alternative approach which overcomes a number of limitations of cell-based therapy. In conclusion, treatment using MSC-CM appears to be highly effective for the treatment of psoriasis and may represent a new avenue of therapy. Further investigations for addressing a number of question provoked by the findings reported here, such as long-term effects (over a period of years), induced changes at cellular and histological levels, and identification of involved bioactive molecules, demand more studies.

Ethical Approval

This study was approved by the Institutional Ethics Committee (approval no. ECR/303/Indt./GJ/2018).

Consent

The patient and the volunteer, participating in the study, were informed about the procedures and their consent was obtained in advance.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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