Autologous bone marrow derived mesenchymal stem cells are safe for the treatment of Achilles tendinopathy.

Achilles tendinopathy is a disabling condition that affects more than 50% of runners. Pre-clinical studies in a large animal model of naturally-occurring tendinopathy similar to human Achilles tendinopathy has shown benefits of autologous bone marrow-derived mesenchymal stem cell (MSC) implantation. However, MSCs are advanced therapies medicinal products (ATMPs), with strict regulatory requirements. Guided by the regulator we carried out a first in man study to assess the safety and efficacy of autologous MSC injection in human patients with non-insertional Achilles tendinopathy. Ten patients, mean age 47 with mid-portion Achilles tendon pain and swelling for more than 6 months, underwent autologous cultured cell injections (median 12.2 × 106, range 5-19 × 106 cells) into their Achilles tendon. At 24 weeks follow-up, no serious adverse reactions or important medical events were observed. MOXFQ, EQ-5D-5L, and VISA-A scores improved clinically at 12 and 24 weeks. VAS pain improved increasingly at 6, 12 and 24 weeks. MOXFQ Pain and VISA-A Scores improved > 12 points from baseline to 24 weeks in 8 patients. Maximum anteroposterior tendon thickness as measured by greyscale US decreased by mean 0.8 mm at 24 weeks. This phase IIa study demonstrated the safety of autologous MSC injection for non-insertional Achilles tendinopathy and provides proof-of-concept of the technique in patients, all of whom had previously failed conservative treatments for chronic disease and leads the way for a larger randomised controlled trial.

Natural Killer Cell Dysfunction in Premenopausal BRCA1 Mutation Carriers: A Potential Mechanism for Ovarian Carcinogenesis.

BACKGROUND: Tissue-specificity for fimbrial fallopian tube ovarian carcinogenesis remains largely unknown in BRCA1 mutation carriers. We aimed to assess the cell autonomous and cell-nonautonomous implications of a germline BRCA1 mutation in the context of cancer immunosurveillance of CD3- CD56+ natural killer (NK) cells. METHODS: Premenopausal BRCA1 mutation carriers versus age-matched non-carriers were compared. Daily urinary 5ß-pregnanediol levels were used to determine progesterone metabolomics across an ovarian cycle. Using peripherally acquired NK cells the cell-mediated cytotoxicity of tumor targets (OVCAR-3, K-562) was determined using live cellular impedance (xCELLigence®) and multicolor flow cytometry. Hypoxia-inducible factor 1-alpha (HIF-1α) immunohistochemistry of cancer-free fallopian tube specimens allowed a comparison of proximal versus distal portions. Utilizing these findings the role of environmental factors relevant to the fimbrial fallopian tube (progesterone, hypoxia) on NK cell functional activity were studied in an ovarian phase-specific manner. RESULTS: BRCA1 mutation carriers demonstrate a differential progesterone metabolome with a phase-specific reduction of peripheral NK cell functional activity. Progesterone exposure further impairs NK cell-mediated cytotoxicity in a dose-dependent manner, which is reversed with the addition of mifepristone (1.25 µM). The fimbrial fallopian tube demonstrated significantly higher HIF-1α staining, particularly in BRCA1 mutation carriers, reflecting a site-specific 'hypoxic niche'. Exposure to hypoxic conditions (1% O2) can further impair tumor cytotoxicity in high-risk carriers. CONCLUSIONS: Phase-specific differential NK cell activity in BRCA1 mutation carriers, either systemically or locally, may favor site-specific pre-invasive carcinogenesis. These cumulative effects across a reproductive lifecycle in high-risk carriers can have a detrimental effect further supporting epidemiological evidence for ovulation inhibition.

Bringing function to the forefront of cell therapy: how do we demonstrate potency?

Unlike conventional pharmaceuticals, biologics and Advanced Therapy Medicinal Products (ATMPs) are required to meet a standard of "potency" as part of the final release criteria at completion of manufacture. During early phase clinical trials, most regulatory agencies have been willing to accept very immature potency assays with an expectation that these will be improved, qualified and validated during the clinical development of the drug to Marketing Authorisation Application (MAA) or Biologics License Application (BLA) submission.This model of continuous development of potency assay in parallel with drug development has already led to at least two notable problem cases; namely Iovance and Mesoblast. Both companies completed successful phase III clinical trials but, in both cases, the initial BLA was rejected on the basis that their potency assay for drug product release was inadequate. Fortunately these issues appear to have been overcome in March of this year, with Mesoblast receiving acceptance of their BLA for Remestemcel and Iovance obtaining a rolling BLA approval for Lifileucel.

A Killer Disarmed: Natural Killer Cell Impairment in Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) treatment remains a big challenge due to the heterogeneous nature of the disease and its ability to progress to acute myeloid leukemia (AML). The only curative option is allogeneic hematopoietic stem cell transplantation (HSCT), but most patients are unfit for this procedure and are left with only palliative treatment options, causing a big unmet need in the context of this disease. Natural killer (NK) cells are attractive candidates for MDS immunotherapy due to their ability to target myeloid leukemic cells without prior sensitization, and in recent years we have seen an arising number of clinical trials in AML and, recently, MDS. NK cells are reported to be highly dysfunctional in MDS patients, which can be overcome by adoptive NK cell immunotherapy or activation of endogenous NK cells. Here, we review the role of NK cells in MDS, the contribution of the tumor microenvironment (TME) to NK cell impairment, and the most recent data from NK cell-based clinical trials in MDS.

Human platelet lysate enhances proliferation but not chondrogenic differentiation of pediatric mesenchymal progenitors.

BACKGROUND AIMS: Cell therapies have the potential to improve reconstructive procedures for congenital craniofacial cartilage anomalies such as microtia. Adipose-derived stem cells (ADSCs) and auricular cartilage stem/progenitor cells (CSPCs) are promising candidates for cartilage reconstruction, but their successful use in the clinic will require the development of xeno-free expansion and differentiation protocols that can maximize their capacity for chondrogenesis. METHODS: We assessed the behavior of human ADSCs and CSPCs grown either in qualified fetal bovine serum (FBS) or human platelet lysate (hPL), a xeno-free alternative, in conventional monolayer and 3-dimensional spheroid cultures. RESULTS: We show that CSPCs and ADSCs display greater proliferation rate in hPL than FBS and express typical mesenchymal stromal cell surface antigens in both media. When expanded in hPL, both cell types, particularly CSPCs, maintain a spindle-like morphology and lower surface area over more passages than in FBS. Both media supplements support chondrogenic differentiation of CSPCs and ADSCs grown either as monolayers or spheroids. However, chondrogenesis appears less ordered in hPL than FBS, with reduced co-localization of aggrecan and collagen type II in spheroids. CONCLUSIONS: hPL may be beneficial for the expansion of cells with chondrogenic potential and maintaining stemness, but not for their chondrogenic differentiation for tissue engineering or disease modeling.

Lessons learned from pre-clinical testing of xenogeneic decellularized esophagi in a rabbit model.

Decellularization of esophagi from several species for tissue engineering is well described, but successful implantation in animal models of esophageal replacement has been challenging. The purpose of this study was to assess feasibility and applicability of esophageal replacement using decellularized porcine esophageal scaffolds in a new pre-clinical model. Following surgical replacement in rabbits with a vascularizing muscle flap, we observed successful anastomoses of decellularized scaffolds, cues of early neovascularization, and prevention of luminal collapse by the use of biodegradable stents. However, despite the success of the surgical procedure, the long-term survival was limited by the fragility of the animal model. Our results indicate that transplantation of a decellularized porcine scaffold is possible and vascular flaps may be useful to provide a vascular supply, but long-term outcomes require further pre-clinical testing in a different large animal model.

Durable Responses and Low Toxicity After Fast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in Adults With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia.

PURPOSE: Prognosis for adult B-cell acute lymphoblastic leukemia (B-ALL) is poor, and there are currently no licensed CD19 chimeric antigen receptor (CAR) therapeutics. We developed a novel second-generation CD19-CAR (CAT19-41BB-Z) with a fast off rate, designed for more physiologic T-cell activation to reduce toxicity and improve engraftment. We describe the multicenter phase I ALLCAR19 (NCT02935257) study of autologous CAT19-41BB-Z CAR T cells (AUTO1) in relapsed or refractory (r/r) adult B-ALL. METHODS: Patients age ≥ 16 years with r/r B-ALL were eligible. Primary outcomes were toxicity and manufacturing feasibility. Secondary outcomes were depth of response at 1 and 3 months, persistence of CAR-T, incidence and duration of hypogammaglobulinemia and B-cell aplasia, and event-free survival and overall survival at 1 and 2 years. RESULTS: Twenty-five patients were leukapheresed, 24 products were manufactured, and 20 patients were infused with AUTO1. The median age was 41.5 years; 25% had prior blinatumomab, 50% prior inotuzumab ozogamicin, and 65% prior allogeneic stem-cell transplantation. At the time of preconditioning, 45% had ≥ 50% bone marrow blasts. No patients experienced ≥ grade 3 cytokine release syndrome; 3 of 20 (15%) experienced grade 3 neurotoxicity that resolved to ≤ grade 1 within 72 hours with steroids. Seventeen of 20 (85%) achieved minimal residual disease-negative complete response at month 1, and 3 of 17 underwent allogeneic stem-cell transplantation while in remission. The event-free survival at 6 and 12 months was 68.3% (42.4%-84.4%) and 48.3% (23.1%-69.7%), respectively. High-level expansion (Cmax 127,152 copies/µg genomic DNA) and durable CAR-T persistence were observed with B-cell aplasia ongoing in 15 of 20 patients at last follow-up. CONCLUSION: AUTO1 demonstrates a tolerable safety profile, high remission rates, and excellent persistence in r/r adult B-ALL. Preliminary data support further development of AUTO1 as a stand-alone treatment for r/r adult B-ALL.

GMP compliant isolation of mucosal epithelial cells and fibroblasts from biopsy samples for clinical tissue engineering.

Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). The current gold standard for growth of epithelial cells for research utilises growth arrested murine 3T3 J2 feeder layers, which are not available for use as a GMP compliant raw material. Using porcine mucosal tissue, we demonstrate a new method for obtaining and growing non-keratinised squamous epithelial cells and fibroblast cells from a single biopsy, replacing the 3T3 J2 with a growth arrested primary fibroblast feeder layer and using pooled Human Platelet lysate (HPL) as the media serum supplement to replace foetal bovine serum (FBS). The initial isolation of the cells was semi-automated using an Octodissociator and the resultant cell suspension cryopreservation for future use. When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells. Furthermore, this method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet.

Allogeneic Mesenchymal Stromal Cell Injection to Alleviate Ischemic Heart Failure Following Arterial Switch Operation.

Cell therapy is a promising tool to prevent and treat heart failure in congenital heart disease. We report the first case of intramyocardial injection of allogeneic mesenchymal stromal cells as rescue therapy in a neonate with ischemic heart failure following arterial switch procedure for isolated transposition of the great arteries. (Level of Difficulty: Advanced.).

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia.

BACKGROUND: With therapeutic hypothermia (HT) for neonatal encephalopathy, disability rates are reduced, but not all babies benefit. Pre-clinical rodent studies suggest mesenchymal stromal cells (MSCs) augment HT protection. AIMS: The authors studied the efficacy of intravenous (IV) or intranasal (IN) human umbilical cord-derived MSCs (huMSCs) as adjunct therapy to HT in a piglet model. METHODS: A total of 17 newborn piglets underwent transient cerebral hypoxia-ischemia (HI) and were then randomized to (i) HT at 33.5°C 1-13 h after HI (n = 7), (ii) HT+IV huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5). Phosphorus-31 and hydrogen-1 magnetic resonance spectroscopy (MRS) was performed at 30 h and 72 h and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and oligodendrocytes quantified. In two further piglets, 30 × 106 IN PKH-labeled huMSCs were administered. RESULTS: HI severity was similar between groups. Amplitude-integrated electroencephalogram (aEEG) recovery was more rapid for HT+IN huMSCs compared with HT from 25 h to 42 h and 49 h to 54 h (P ≤ 0.05). MRS phosphocreatine/inorganic phosphate was higher on day 2 in HT+IN huMSCs than HT (P = 0.035). Comparing HT+IN huMSCs with HT and HT+IV huMSCs, there were increased OLIG2 counts in hippocampus (P = 0.011 and 0.018, respectively), internal capsule (P = 0.013 and 0.037, respectively) and periventricular white matter (P = 0.15 for IN versus IV huMSCs). Reduced TUNEL-positive cells were seen in internal capsule with HT+IN huMSCs versus HT (P = 0.05). PKH-labeled huMSCs were detected in the brain 12 h after IN administration. CONCLUSIONS: After global HI, compared with HT alone, the authors saw beneficial effects of HT+IN huMSCs administered at 24 h and 48 h (30 × 106 cells/kg total dose) based on more rapid aEEG recovery, improved 31P MRS brain energy metabolism and increased oligodendrocyte survival at 72 h.

Killers at the crossroads: The use of innate immune cells in adoptive cellular therapy of cancer.

Adoptive cell therapy (ACT) is an approach to cancer treatment that involves the use of antitumor immune cells to target residual disease in patients after completion of chemo/radiotherapy. ACT has several advantages compared with other approaches in cancer immunotherapy, including the ability to specifically expand effector cells in vitro before selection for adoptive transfer, as well as the opportunity for host manipulation in order to enhance the ability of transferred cells to recognize and kill established tumors. One of the main challenges to the success of ACT in cancer clinical trials is the identification and generation of antitumor effector cells with high avidity for tumor recognition. Natural killer (NK) cells, cytokine-induced killers and natural killer T cells are key innate or innate-like effector cells in cancer immunosurveillance that act at the interface between innate and adaptive immunity, to have a greater influence over immune responses to cancer. In this review, we discuss recent studies that highlight their potential in cancer therapy and summarize clinical trials using these effector immune cells in adoptive cellular therapy for the treatment of cancer.

Tumor- and cytokine-primed human natural killer cells exhibit distinct phenotypic and transcriptional signatures.

An emerging cellular immunotherapy for cancer is based on the cytolytic activity of natural killer (NK) cells against a wide range of tumors. Although in vitro activation, or "priming," of NK cells by exposure to pro-inflammatory cytokines, such as interleukin (IL)-2, has been extensively studied, the biological consequences of NK cell activation in response to target cell interactions have not been thoroughly characterized. We investigated the consequences of co-incubation with K562, CTV-1, Daudi RPMI-8226, and MCF-7 tumor cell lines on the phenotype, cytokine expression profile, and transcriptome of human NK cells. We observe the downregulation of several activation receptors including CD16, CD62L, C-X-C chemokine receptor (CXCR)-4, natural killer group 2 member D (NKG2D), DNAX accessory molecule (DNAM)-1, and NKp46 following tumor-priming. Although this NK cell phenotype is typically associated with NK cell dysfunction in cancer, we reveal the upregulation of NK cell activation markers, such as CD69 and CD25; secretion of pro-inflammatory cytokines, including macrophage inflammatory proteins (MIP-1) α /ß and IL-1ß/6/8; and overexpression of numerous genes associated with enhanced NK cell cytotoxicity and immunomodulatory functions, such as FAS, TNFSF10, MAPK11, TNF, and IFNG. Thus, it appears that tumor-mediated ligation of receptors on NK cells may induce a primed state which may or may not lead to full triggering of the lytic or cytokine secreting machinery. Key signaling molecules exclusively affected by tumor-priming include MAP2K3, MARCKSL1, STAT5A, and TNFAIP3, which are specifically associated with NK cell cytotoxicity against tumor targets. Collectively, these findings help define the phenotypic and transcriptional signature of NK cells following their encounters with tumor cells, independent of cytokine stimulation, and provide insight into tumor-specific NK cell responses to inform the transition toward harnessing the therapeutic potential of NK cells in cancer.

Characterization of human natural killer cells for therapeutic use.

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.

T-cell receptor gene-modified cells: past promises, present methodologies and future challenges.

Immunotherapy constitutes an exciting and rapidly evolving field, and the demonstration that genetically modified T-cell receptors (TCRs) can be used to produce T-lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. Overall, TCR-modified T cells have the ability to target a wide variety of self and non-self targets through the normal biology of a T cell. Although major histocompatibility complex (MHC)-restricted and dependent on co-receptors, genetically engineered TCRs still present a number of characteristics that ensure they are an important alternative strategy to chimeric antigen receptors (CARs), and high-affinity TCRs can now be successfully engineered with the potential to enhance therapeutic efficacy while minimizing adverse events. This review will focus on the main characteristics of TCR gene-modified cells, their potential clinical application and promise to the field of adoptive cell transfer (ACT), basic manufacturing procedures and characterization protocols and overall challenges that need to be overcome so that redirection of TCR specificity may be successfully translated into clinical practice, beyond early-phase clinical trials.

Silsesquioxane polymer as a potential scaffold for laryngeal reconstruction.

Cancer, disease and trauma to the larynx and their treatment can lead to permanent loss of structures critical to voice, breathing and swallowing. Engineered partial or total laryngeal replacements would need to match the ambitious specifications of replicating functionality, outer biocompatibility, and permissiveness for an inner mucosal lining. Here we present porous polyhedral oligomeric silsesquioxane-poly(carbonate urea) urethane (POSS-PCUU) as a potential scaffold for engineering laryngeal tissue. Specifically, we employ a precipitation and porogen leaching technique for manufacturing the polymer. The polymer is chemically consistent across all sample types and produces a foam-like scaffold with two distinct topographies and an internal structure composed of nano- and micro-pores. While the highly porous internal structure of the scaffold contributes to the complex tensile behaviour of the polymer, the surface of the scaffold remains largely non-porous. The low number of pores minimise access for cells, although primary fibroblasts and epithelial cells do attach and proliferate on the polymer surface. Our data show that with a change in manufacturing protocol to produce porous polymer surfaces, POSS-PCUU may be a potential candidate for overcoming some of the limitations associated with laryngeal reconstruction and regeneration.

A Phase 1 Trial of CNDO-109-Activated Natural Killer Cells in Patients with High-Risk Acute Myeloid Leukemia.

Natural killer (NK) cells are an emerging immunotherapy approach to acute myeloid leukemia (AML); however, the optimal approach to activate NK cells before adoptive transfer remains unclear. Human NK cells that are primed with the CTV-1 leukemia cell line lysate CNDO-109 exhibit enhanced cytotoxicity against NK cell-resistant cell lines. To translate this finding to the clinic, CNDO-109-activated NK cells (CNDO-109-NK cells) isolated from related HLA-haploidentical donors were evaluated in a phase 1 dose-escalation trial at doses of 3 × 105 (n = 3), 1 × 106 (n = 3), and 3 × 106 (n = 6) cells/kg in patients with AML in first complete remission (CR1) at high risk for recurrence. Before CNDO-109-NK cell administration, patients were treated with lymphodepleting fludarabine/cyclophosphamide. CNDO-109-NK cells were well tolerated, and no dose-limiting toxicities were observed at the highest tested dose. The median relapse-free survival (RFS) by dose level was 105 (3 × 105), 156 (1 × 106), and 337 (3 × 106) days. Two patients remained relapse-free in post-trial follow-up, with RFS durations exceeding 42.5 months. Donor NK cell microchimerism was detected on day 7 in 10 of 12 patients, with 3 patients having evidence of donor cells on day 14 or later. This trial establishes that CNDO-109-NK cells generated from related HLA haploidentical donors, cryopreserved, and then safely administered to AML patients with transient persistence without exogenous cytokine support. Three durable complete remissions of 32.6 to 47.6+ months were observed, suggesting additional clinical investigation of CNDO-109-NK cells for patients with myeloid malignancies, alone or in combination with additional immunotherapy strategies, is warranted.

The survey on cellular and tissue-engineered therapies in Europe and neighboring Eurasian countries in 2014 and 2015.

BACKGROUND AIMS: With the support of five established scientific organizations, this report, the seventh of its kind, describes activity in Europe for the years 2014 and 2015 in the area of cellular and tissue-engineered therapies, excluding hematopoietic stem cell (HSC) treatments for the reconstitution of hematopoiesis. METHODS: In 2015 [respectively 2014], 205 [276] teams from 32 countries responded to the cellular and tissue-engineered therapy survey; 178 [126] teams reported treating 3686 [2665] patients. RESULTS: Indications were musculoskeletal/rheumatological disorders (32% [33%]), cardiovascular disorders (12% [21%]), hematology/oncology (predominantly prevention or treatment of graft versus host disease and HSC graft enhancement; 20% [20%]), neurological disorders (4% [6%]), gastrointestinal disorders (<1% [1%]) and other indications (31% [20%]). The majority of autologous cells (60% [73%]) were used to treat musculoskeletal/rheumatological (44% [36%]) disorders, whereas allogeneic cells were used mainly for hematology/oncology (61% [68%]). The reported cell types were mesenchymal stromal cells (40% [49%]), chondrocytes (13% [6%]), hematopoietic stem cells (12% [23%]), dermal fibroblasts (8% [3%]), dendritic cells (2% [2%]), keratinocytes (1% [2%]) and others (24% [15%]). Cells were expanded in vitro in 63% [40%] of the treatments, sorted in 16% [6%] of the cases and rarely transduced (<1%). Cells were delivered predominantly as suspension 43% [51%], intravenously or intra-arterially (30% [30%]), or using a membrane/scaffold (25% [19%]). DISCUSSION: The data are compared with those from previous years to identify trends in a still unpredictably evolving field. Perspectives of representatives from plastic surgery practitioners, Iran and ISCT are presented (contributing authors D.A. Barbara, B. Hossein and W.L. Mark, respectively).

Autologous Cell Seeding in Tracheal Tissue Engineering.

PURPOSE OF REVIEW: There is no consensus on the best technology to be employed for tracheal replacement. One particularly promising approach is based upon tissue engineering and involves applying autologous cells to transplantable scaffolds. Here, we present the reported pre-clinical and clinical data exploring the various options for achieving such seeding. RECENT FINDINGS: Various cell combinations, delivery strategies, and outcome measures are described. Mesenchymal stem cells (MSCs) are the most widely employed cell type in tracheal bioengineering. Airway epithelial cell luminal seeding is also widely employed, alone or in combination with other cell types. Combinations have thus far shown the greatest promise. Chondrocytes may improve mechanical outcomes in pre-clinical models, but have not been clinically tested. Rapid or pre-vascularization of scaffolds is an important consideration. Overall, there are few published objective measures of post-seeding cell viability, survival, or overall efficacy. SUMMARY: There is no clear consensus on the optimal cell-scaffold combination and mechanisms for seeding. Systematic in vivo work is required to assess differences between tracheal grafts seeded with combinations of clinically deliverable cell types using objective outcome measures, including those for functionality and host immune response.

Assessing cell-nanoparticle interactions by high content imaging of biocompatible iron oxide nanoparticles as potential contrast agents for magnetic resonance imaging.

Stem cell tracking in cellular therapy and regenerative medicine is an urgent need, superparamagnetic iron oxide nanoparticles (IONPs) could be used as contrast agents in magnetic resonance imaging (MRI) that allows visualization of the implanted cells ensuring they reach the desired sites in vivo. Herein, we report the study of the interaction of 3,4-dihydroxyhydrocinnamic acid (DHCA) functionalized IONPs that have desirable properties for T2 - weighted MRI, with bone marrow-derived primary human mesenchymal stem cells (hMSCs). Using the multiparametric high-content imaging method, we evaluate cell viability, formation of reactive oxygen species, mitochondrial health, as well as cell morphology and determine that the hMSCs are minimally affected after labelling with IONPs. Their cellular uptake is visualized by transmission electron microscopy (TEM) and Prussian Blue staining, and quantified using an iron specific colourimetric method. In vitro and in vivo studies demonstrate that these IONPs are biocompatible and can produce significant contrast enhancement in T2-weighted MRI. Iron oxide nanoparticles are detected in vivo as hypointense regions in the liver up to two weeks post injection using 9.4 T MRI. These DHCA functionalized IONPs are promising contrast agents for stem cell tracking by T2-weighted MRI as they are biocompatible and show no evidence of cytotoxic effects on hMSCs.

Tracheal Replacement Therapy with a Stem Cell-Seeded Graft: Lessons from Compassionate Use Application of a GMP-Compliant Tissue-Engineered Medicine.

Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials. Stem Cells Translational Medicine 2017;6:1458-1464.