Harmonised culture procedures minimise but do not eliminate mesenchymal stromal cell donor and tissue variability in a decentralised multicentre manufacturing approach.

BACKGROUND: Mesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A- to assess the impact on MSC characteristics when different laboratories, performed analysis on the same MSC material using harmonised culture conditions and B- to understand source-specific differences. METHODS: Three independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue-specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). RESULTS: By implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. CONCLUSION: These results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of harmonised culture procedures can reduce but do not eliminate inter-lab and operator differences.

Fate of intravenously administered umbilical cord mesenchymal stromal cells and interactions with the host's immune system.

Mesenchymal stromal cells (MSCs) are multipotent cells showing promise in pre-clinical studies and currently used in many clinical trials. The regenerative potential of MSCs is mediated, at least in part, by direct and indirect immunomodulatory processes. However, the mechanism of action is not fully understood yet, and there are still concerns about possible undesired negative effects associated with the administration of living cells. In this study, we (i) compare the long-term fate and safety of umbilical cord (UC-)MSCs administered to immunocompetent and immunocompromised (severe combined immunodeficient (SCID) and non-obese diabetic (NOD)/SCID) animals, and (ii) investigate the immunological response of the host to the administered cells. Intravenous administration of firefly luciferase expressing UC-MSCs revealed that the cells get trapped in the lungs of both immunocompetent and immunocompromised animals, with > 95% of the cells disappearing within 72 h after administration. In 27% of the SCID and 45% of the NOD/SCID, a small fraction of the cells lived up to day 14 but in most cases they all disappeared earlier. One NOD/SCID mouse showed a weak signal up to day 31. Immunocompetent mice displayed elevated percentages of neutrophils in the lungs, the blood, and the spleen 2 h after the administration of the cells. The concentration of neutrophil chemoattractants (MCP1, CCL7, Gro-α and IP-10) were also increased in the plasma of the animals 2 h after the administration of the MSCs. Our results suggest that although the UC-MSCs are short-lived in mice, they still result in an immunological response that might contribute to a therapeutic effect.

DEAE-Dextran Enhances the Lentiviral Transduction of Primary Human Mesenchymal Stromal Cells from All Major Tissue Sources Without Affecting Their Proliferation and Phenotype.

Genetic engineering of mesenchymal stromal cells (MSCs) is a tool widely used to explore MSC properties in vitro and in vivo. Lentiviral infection with the use of polycations as an adjuvant is a method that is commonly used to generate stably transduced cells. However, it is known that some polycations can negatively affect primary MSCs and to date, no study has explored the effect of different polycations on the transduction efficiency and properties of all main types of MSCs, namely those derived from umbilical cord, bone marrow and adipose tissue. Here we explore a range of polycations, using transduction protocols with and without spinoculation, to produce stably transduced MSCs from these three tissue sources. We identified that an overnight incubation with diethylaminoethyl-dextran (DEAE-Dextran) is the protocol associated with the best transduction efficiency without compromising the viability of the cells, and which worked consistently with lentiviral particles encoding for different transgenes. Transduced and sorted MSC populations revealed no significant changes in proliferation, morphology and expression of MSC markers compared to naïve MSCs. Following this study, we conclude that DEAE-Dextran is a polycation that can be successfully used to enhance the transduction of MSCs from all major tissue sources.

Firefly luciferase offers superior performance to AkaLuc for tracking the fate of administered cell therapies.

INTRODUCTION: A novel, red-shifted bioluminescence imaging (BLI) system called AkaBLI has been recently developed for cell tracking in preclinical models and to date, limited data is available on how it performs in relation to existing systems. PURPOSE: To systematically compare the performance of AkaBLI and the standard Firefly luciferase (FLuc) systems to monitor the biodistribution and fate of cell therapies in rodents. METHODS: Umbilical cord mesenchymal stromal cells (MSCs) were transduced to produce two genetically engineered populations, expressing either AkaLuc or the engineered FLuc luc2. The bioluminescence of AkaLuc+ and FLuc+ cells was assessed both in vitro (emission spectra, saturation kinetics and light emission per cell) and in vivo (substrate kinetics following intraperitoneal and subcutaneous administration and biodistribution of the cells up to day 7). RESULTS: Introduction of the reporter genes has no effect on MSC phenotype. For BLI, the FLuc system is superior to AkaBLI in terms of (i) light output, producing a stronger signal after subcutaneous substrate delivery and more consistent signal kinetics when delivered intraperitoneally; (ii) absence of hepatic background; and (iii) safety, where the AkaLuc substrate was associated with a reaction in the skin of the mice in vivo. CONCLUSION: We conclude that there is no advantage in using the AkaBLI system to track the biodistribution of systemically administered cell-based regenerative medicine therapies in vivo.

From cancer screening to treatment: service delivery and referral in the National Breast and Cervical Cancer Early Detection Program.

The National Breast and Cervical Cancer Early Detection Program (NBCCEDP) provides breast and cervical cancer screening and diagnostic services to low-income and underserved women through a network of providers and health care organizations. Although the program serves women 40-64 years old for breast cancer screening and 21-64 years old for cervical cancer screening, the priority populations are women 50-64 years old for breast cancer and women who have never or rarely been screened for cervical cancer. From 1991 through 2011, the NBCCEDP provided screening and diagnostic services to more than 4.3 million women, diagnosing 54,276 breast cancers, 2554 cervical cancers, and 123,563 precancerous cervical lesions. A critical component of providing screening services is to ensure that all women with abnormal screening results receive appropriate and timely diagnostic evaluations. Case management is provided to assist women with overcoming barriers that would delay or prevent follow-up care. Women diagnosed with cancer receive treatment through the states' Breast and Cervical Cancer Treatment Programs (a special waiver for Medicaid) if they are eligible. The NBCCEDP has performance measures that serve as benchmarks to monitor the completeness and timeliness of care. More than 90% of the women receive complete diagnostic care and initiate treatment less than 30 days from the time of their diagnosis. Provision of effective screening and diagnostic services depends on effective program management, networks of providers throughout the community, and the use of evidence-based knowledge, procedures, and technologies.

Differential role of Th1 and Th2 cytokines in autotoxicity driven by CD19-specific second-generation chimeric antigen receptor T cells in a mouse model.

T cells engrafted with chimeric AgRs (CAR) are showing exciting potential for targeting B cell malignancies in early-phase clinical trials. To determine whether the second-generation CAR was essential for optimal antitumor activity, two CD28-based CAR constructs targeting CD19 were tested for their ability to redirect mouse T cell function against established B cell lymphoma in a BALB/c syngeneic model system. T cells armed with either CAR eliminated A20 B cell lymphoma in vivo; however, one construct induced a T cell dose-dependent acute toxicity associated with a raised serum Th1 type cytokine profile on transfer into preconditioned mice. Moreover, a chronic toxicity manifested as granuloma-like formation in spleen, liver, and lymph nodes was observed in animals receiving T cells bearing either CD28 CAR, albeit with different kinetics dependent upon the specific receptor used. This phenotype was associated with an expansion of CD4+ CAR+ T cells and CD11b+ Gr-1(+) myeloid cells and increased serum Th2-type cytokines, including IL-10 and IL-13. Mouse T cells engrafted with a first-generation CAR failed to develop such autotoxicity, whereas toxicity was not apparent when T cells bearing the same receptors were transferred into C57BL/6 or C3H animals. In summary, the adoptive transfer of second-generation CD19-specific CAR T cells can result in a cell dose-dependent acute toxicity, whereas the prolonged secretion of high levels of Th2 cytokines from these CAR T cells in vivo drives a granulomatous reaction resulting in chronic toxicity. Strategies that prevent a prolonged Th2-cytokine biased CAR T cell response are clearly warranted.

CAR T cells: driving the road from the laboratory to the clinic.

Blockbuster antibody therapies have catapulted immune-based approaches to treat cancer into the consciousness of mainstay clinical research. On the back of this, other emerging immune-based therapies are providing great promise. T-cell therapy is one such area where recent trials using T cells genetically modified to express an antibody-based chimeric antigen receptor (CAR) targeted against the CD19 antigen have demonstrated impressive responses when adoptively transferred to patients with advanced chronic lymphocytic leukemia. The general concept of the CAR T cell was devised some 20 years ago. In this relatively short period of time, the technology to redirect T-cell function has moved at pace facilitating clinical translation; however, many questions remain with respect to developing the approach to improve CAR T-cell therapeutic activity and also to broaden the range of tumors that can be effectively targeted by this approach. This review highlights some of the underlying principles and compromises of CAR T-cell technology using the CD19-targeted CAR as a paradigm and discusses some of the issues that relate to targeting solid tumors with CAR T cells.

Number of cervical biopsies and sensitivity of colposcopy.

OBJECTIVE: To examine the influence that type of medical training and number of biopsies have on sensitivity of colposcopically guided biopsies. METHODS: Among 408 women with an adequate enrollment colposcopy and a diagnosis of cervical intraepithelial neoplasia (CIN) 3 or cancer (CIN 3+) over 2 years in the Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesions (ASCUS-LSIL) Triage Study, we evaluated factors influencing the sensitivity of the enrollment colposcopic procedure. We used contingency table analysis to examine confounding variables and chi(2) tests to ascertain statistical significance. RESULTS: Overall, 69.9% of women with a cumulative diagnosis of CIN 3+ had a "true-positive" enrollment colposcopically guided biopsy result of CIN 2 or worse (CIN 2+), the threshold that would trigger excisional therapy. The sensitivity of the procedure did not vary significantly by type of colposcopist. However, the sensitivity was significantly greater when the colposcopists took two or more biopsies instead of one (P<.01), a pattern observed across all types of colposcopists. Independent of the severity of the colposcopic impression, the frequency with which colposcopists took two or more biopsies instead of one varied (in descending order) from nurse practitioners to general gynecologists to gynecologic oncology fellows to gynecologic oncologists (P<.01). CONCLUSION: Colposcopy with guided biopsy or biopsies detects approximately two thirds of CIN 3+. Although the sensitivity of the procedure does not differ significantly by type of medical training, it is greater when two or more biopsies are taken.

ASCCP patient management guidelines: Pap test specimen adequacy and quality indicators.

Our objective was to provide management guidelines according to Papanicolaou (Pap) test specimen adequacy based on literature review and expert opinion. A task force named by the American Society for Colposcopy and Cervical Pathology (ASCCP) conducted a literature review and discussed appropriate management. The Steering Committee of the ASCCP and other experts reviewed the guidelines. The guidelines recommend a repeated Pap test in 12 months for most women undergoing routine annual/biennial screening if the current Pap test is negative but either lacks an endocervical/ transformation zone component or is partially obscured. Indications for considering an earlier repeat are also provided. The preferred managementfor unsatisfactory Pap tests is a repeated Pap test within a short interval of 2 to 4 months. The management guidelines will help promote optimal and uniform follow-up of women according to Pap test specimen adequacy.

ASCCP Patient Management Guidelines: Pap Test Specimen Adequacy and Quality Indicators.

OBJECTIVES: To provide management guidelines according to Pap test specimen adequacy based on literature review and expert opinion. METHODS: A task force named by the American Society for Colposcopy and Cervical Pathology conducted a literature review and discussed appropriate management. The steering committee of the American Society for Colposcopy and Cervical Pathology and other experts reviewed the guidelines. RESULTS: The guidelines recommend a repeat Pap test in 12 months for most women who are undergoing routine annual/biennial screening if the current Pap test is negative but either lacks an endocervical/transformation zone component or is partially obscured. Indications for considering an earlier repeat are also provided. The preferred management for unsatisfactory Pap tests is a repeat Pap test within a short interval of 2 to 4 months. CONCLUSIONS: The management guidelines will help promote optimal and uniform follow-up of women's Pap tests, according to Pap test specimen adequacy.