Selection and validation of reference genes for qPCR analysis of differentiation and maturation of THP-1 cells into M1 macrophage-like cells.

For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ), after which it differentiates and polarizes into proinflammatory M1-like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription-quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP-1 cells stimulated with LPS and IFN-γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), UBC (ubiquitin C), B2M (0ß2-microbulin), ACTB (ß-actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP-1 monocyte model system.

The Initial Cardiac Tissue Response to Cryopreserved Allogeneic Adipose Tissue-Derived Mesenchymal Stromal Cells in Rats with Chronic Ischemic Cardiomyopathy.

Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.

Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate.

In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95 × 106 cells were suspended in 10% FBS or 5% hPL medium, and loaded into a bioreactor coated with cryoprecipitate. ASCs (P0) were harvested, and 30 × 106 ASCs were reloaded for continued expansion (P1). Feeding rate and time of harvest was guided by metabolic monitoring. Viability, sterility, purity, differentiation capacity, and genomic stability of ASCs P1 were determined. Cultivation of SVF in hPL medium for in average nine days, yielded 546 × 106 ASCs compared to 111 × 106 ASCs, after 17 days in FBS medium. ASCs P1 yields were in average 605 × 106 ASCs (PD [population doublings]: 4.65) after six days in hPL medium, compared to 119 × 106 ASCs (PD: 2.45) in FBS medium, after 21 days. ASCs fulfilled ISCT criteria and demonstrated genomic stability and sterility. The use of hPL as a growth supplement for ASCs expansion in the quantum cell expansion system provides an efficient expansion process compared to the use of FBS, while maintaining cell quality appropriate for clinical use. The described process is an obvious choice for manufacturing of large-scale allogeneic ASC products.

Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia.

BACKGROUND AIMS: Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. METHODS: ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and ß-galactosidase activity. RESULTS: hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. CONCLUSION: Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions.

Culture expansion of adipose derived stromal cells. A closed automated Quantum Cell Expansion System compared with manual flask-based culture.

BACKGROUND: Adipose derived stromal cells (ASCs) are a rich and convenient source of cells for clinical regenerative therapeutic approaches. However, applications of ASCs often require cell expansion to reach the needed dose. In this study, cultivation of ASCs from stromal vascular fraction (SVF) over two passages in the automated and functionally closed Quantum Cell Expansion System (Quantum system) is compared with traditional manual cultivation. METHODS: Stromal vascular fraction was isolated from abdominal fat, suspended in α-MEM supplemented with 10% Fetal Bovine Serum and seeded into either T75 flasks or a Quantum system that had been coated with cryoprecipitate. The cultivation of ASCs from SVF was performed in 3 ways: flask to flask; flask to Quantum system; and Quantum system to Quantum system. In all cases, quality controls were conducted for sterility, mycoplasmas, and endotoxins, in addition to the assessment of cell counts, viability, immunophenotype, and differentiation potential. RESULTS: The viability of ASCs passage 0 (P0) and P1 was above 96%, regardless of cultivation in flasks or Quantum system. Expression of surface markers and differentiation potential was consistent with ISCT/IFATS standards for the ASC phenotype. Sterility, mycoplasma, and endotoxin tests were consistently negative. An average of 8.0 × 107 SVF cells loaded into a Quantum system yielded 8.96 × 107 ASCs P0, while 4.5 × 106 SVF cells seeded per T75 flask yielded an average of 2.37 × 106 ASCs-less than the number of SVF cells seeded. ASCs P1 expanded in the Quantum system demonstrated a population doubling (PD) around 2.2 regardless of whether P0 was previously cultured in flasks or Quantum, while ASCs P1 in flasks only reached a PD of 1.0. CONCLUSION: Manufacturing of ASCs in a Quantum system enhances ASC expansion rate and yield significantly relative to manual processing in T-flasks, while maintaining the purity and quality essential to safe and robust cell production. Notably, the use of the Quantum system entails significantly reduced working hours and thereby costs.

Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue.

BACKGROUND: The utility of mesenchymal stromal cells (MSCs) in therapeutic applications for regenerative medicine has gained much attention. Clinical translation of MSC-based approaches requires in vitro culture-expansion to achieve a sufficient number of cells. The ideal cell culture medium should be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. METHODS: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three different commercially available hPL fulfilling good manufacturing practice criteria for clinical use. BMSCs and ASCs cultured in Minimum Essential Medium Eagle-alpha supplemented with 5% PLT-Max (Mill Creek), Stemulate™ PL-S and Stemulate™ PL-SP (COOK General Biotechnology) were compared to standard culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. RESULTS: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS medium. In general, the immunophenotype of both BMSCs and ASCs fulfilled International Society for Cellular Therapy (ISCT) criteria after hPL media expansion. Comparative genomic hybridization measurements demonstrated no unbalanced chromosomal rearrangements for BMSCs or ASCs cultured in hPL media or FBS medium. The BMSCs and ASCs could differentiate into osteogenic, adipogenic, or chondrogenic lineages in all four culture conditions. CONCLUSION: All three clinically approved commercial human platelet lysates accelerated proliferation of BMSCs and ASCs and the cells meet the ISCT mesenchymal phenotypic requirements without exhibiting chromosomal aberrations.

Human adipose-derived stromal cells in a clinically applicable injectable alginate hydrogel: Phenotypic and immunomodulatory evaluation.

BACKGROUND AIMS: Clinical trials have documented beneficial effects of mesenchymal stromal cells from bone marrow and adipose tissue (ASCs) as treatment in patients with ischemic heart disease. However, retention of transplanted cells is poor. One potential way to increase cell retention is to inject the cells in an in situ cross-linked alginate hydrogel. METHODS: ASCs from abdominal human tissue were embedded in alginate hydrogel and alginate hydrogel modified with Arg-Gly-Asp motifs (RGD-alginate) and cultured for 1 week. Cell viability, phenotype, immunogenicity and paracrine activity were determined by confocal microscopy, dendritic cell co-culture, flow cytometry, reverse transcriptase quantitative polymerase chain reaction, Luminex multiplex, and lymphocyte proliferation experiments. RESULTS: ASCs performed equally well in alginate and RGD-alginate. After 1 week of alginate culture, cell viability was >93%. Mesenchymal markers CD90 and CD29 were reduced compared with International Society for Cellular Therapy criteria. Cells sedimented from the alginates during cultivation regained the typical level of these markers, and trilineage differentiation was performed by standard protocols. Hepatocyte growth factor mRNA was increased in ASCs cultivated in alginates compared with monolayer controls. Alginates and alginates containing ASCs did not induce dendritic cell maturation. ASCs in alginate responded like controls to interferon-gamma stimulation (licensing), and alginate culture increased the ability of ASCs to inhibit lymphocyte proliferation. DISCUSSION: ASCs remain viable in alginates; they transiently change phenotype in alginate hydrogel but regain the phenotype of monolayer controls upon release. Cells maintain their paracrine potential while in alginates; the combination of ASCs and alginate is non-immunogenic and, in fact, immunosuppressive.

Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes.

Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

Investigating the paracrine and juxtacrine abilities of adipose-derived stromal cells in angiogenesis triple cell co-cultures.

The pro-angiogenic abilities of adipose-derived stromal cells (ASCs) make them attractive candidates for cellular therapy, especially for ischemic disease indications. However, details regarding the underlying mechanisms remain elusive. Therefore, this study aimed to investigate paracrine and juxtacrine abilities of ASCs in angiogenesis triple cell co-cultures by detailed image analysis of the vascular-like structures. Fibroblast-endothelial cell co-cultures were established, and ASCs were added directly or indirectly through inserts. The cultures were treated with antibodies or subjected to analyses using ELISA and RT2 PCR Arrays. The model consistently generated vascular-like structures. ASCs increased the total branch lengths equally well in paracrine and juxtacrine conditions, by increasing the number of branches and average branch lengths (ABL). In contrast, addition of VEGF to the model increased the number of branches, but not the ABL. Still, ASCs increased the VEGF levels in supernatants of paracrine and juxtacrine co-cultures, and anti-VEGF treatment decreased the sprouting. ASCs themselves up-regulated collagen type V in response to paracrine signals from the co-cultures. The results suggest that ASCs initiate sprouting through secretion of several paracrine factors, among which VEGF is identified, but VEGF alone does not recapitulate the paracrine actions of ASCs. By employing neutralizing antibodies and dismantling common model outputs using image analysis, the triple cell co-culture is an attractive tool for discovery of the paracrine factors in ASCs' secretome which act in concert with VEGF to improve angiogenesis.

Mesenchymal stromal cells to treat patients with non-ischaemic heart failure: Results from SCIENCE II pilot study.

AIMS: Allogeneic stem cell therapy is more logistically suitable compared with autologous cell therapy for large-scale patient treatment. We aim to investigate the clinical safety and efficacy profile of the allogeneic adipose tissue derived mesenchymal stromal cell product (CSCC_ASC) as an add-on therapy in patients with chronic non-ischaemic heart failure with reduced left ventricular ejection fraction (HFrEF) < 40%. METHODS AND RESULTS: This is a single-centre investigator-initiated randomized phase I/II study with direct intra-myocardial injections of 100 million allogeneic CSCC_ASC. A total of 30 HFrEF patients with New York Heart Association (NYHA) class ≥II despite optimal anticongestive heart failure medication and plasma NT-proBNP > 300 pg/mL (>35 pmol/L) were included and randomized 2:1 to CSCC_ASC or standard care. The primary endpoint left ventricular end systolic volume (LVESV) and other echo related parameters were analysed by an investigator blinded for treatment allocation. No difference in serious adverse events was observed between groups. LVESV decreased significantly from baseline to 6 months follow-up in the ASC group (153.7 ± 53.2 mL and 128.7 ± 45.6 mL, P < 0.001) and remained unchanged in the standard care group (180.4 ± 39.4 mL and 186.7 ± 48.9 mL, P = 0.652). There was a significant difference between the groups in LVESV change (31.3 ± 11.0 mL, P = 0.009). The difference from baseline to follow-up between the two groups in left ventricular end diastolic volume (LVEDV) was 18.7 ± 12.4 mL, P = 0.146 and in left ventricular ejection fraction (LVEF) -7.8 ± 2.1%, P = 0.001. Considering the baseline values of LVESV, LVEDV and LVEF as covariates, the difference between groups for change from baseline to follow-up resulted in a P-value of 0.056, 0.076, and 0.738, respectively. NYHA class and self-reported health did also improve significantly in the ASC group compared with the standard care group (0.7 ± 0.2, P = 0.001 and -12.8 ± 5.3, P = 0.025; respectively). There was no difference in NT-proBNP (-371 ± 455 pmol/L, P = 0.422) or in 6 min walk test (12 ± 31 m, P = 0.695) between groups. CONCLUSIONS: Intramyocardial injections of allogeneic CSCC_ASC in patients with chronic non-ischaemic HFrEF was safe and improved LVESV, LVEF, NYHA class, and self-reported health compared with standard care group.

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 and [64Cu]Cu-DOTATATE PET Predict Improvement in Ischemic Cardiomyopathy.

An increasing number of patients are living with chronic ischemic cardiomyopathy (ICM) and/or heart failure. Treatment options and prognostic tools are lacking for many of these patients. Our aim was to investigate the prognostic value of imaging angiogenesis and macrophage activation via positron emission tomography (PET) in terms of functional improvement after cell therapy. Myocardial infarction was induced in rats. Animals were scanned with [18F]FDG PET and echocardiography after four weeks and randomized to allogeneic adipose tissue-derived stromal cells (ASCs, n = 18) or saline (n = 9). Angiogenesis and macrophage activation were assessed before and after treatment by [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE. There was no overall effect of the treatment. Rats that improved left ventricular ejection fraction (LVEF) had higher uptake of both [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE at follow-up (p = 0.006 and p = 0.008, respectively). The uptake of the two tracers correlated with each other (r = 0.683, p = 0.003 pre-treatment and r = 0.666, p = 0.004 post-treatment). SUVmax at follow-up could predict improvement in LVEF (p = 0.016 for [68Ga]Ga-RGD and p = 0.045 for [64Cu]Cu-DOTATATE). High uptake of [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE PET after injection of ASCs or saline preceded improvement in LVEF. The use of these tracers could improve the monitoring of heart failure patients in treatment.

Danish phase II trial using adipose tissue derived mesenchymal stromal cells for patients with ischaemic heart failure.

AIMS: Patients suffering from chronic ischaemic heart failure with reduced left ventricular ejection fraction (HFrEF) have reduced quality-of-life, repetitive hospital admissions, and reduced life expectancy. Allogeneic cell therapy is currently investigated as a potential treatment option after initially encouraging results from clinical autologous and allogeneic trials in patients with HFrEF. We aimed to investigate the allogeneic Cardiology Stem Cell Centre Adipose tissue derived mesenchymal Stromal Cell product (CSCC_ASC) as an add-on therapy in patients with chronic HFrEF. METHODS AND RESULTS: This is a Danish multi-centre double-blinded placebo-controlled phase II study with direct intra-myocardial injections of allogeneic CSCC_ASC. A total of 81 HFrEF patients were included and randomized 2:1 to CSCC_ASC or placebo injections. The inclusion criteria were reduced left ventricular ejection fraction (LVEF ≤ 45%), New York Heart Association (NYHA) class II-III despite optimal anti-congestive heart failure medication and no further revascularization options. Injections of 0.3 mL CSCC_ASC (total cell dose 100 × 106 ASCs) (n = 54) or isotonic saline (n = 27) were performed into the viable myocardium in the border zone of infarcted tissue using the NOGA Myostar® catheter (Biological Delivery System, Cordis, Johnson & Johnson, USA). The primary endpoint, left ventricular end systolic volume (LVESV), was evaluated at 6-month follow-up. The safety was measured during a 3-years follow-up period. RESULTS: Mean age was 67.0 ± 9.0 years and 66.6 ± 8.1 years in the ASC and placebo groups, respectively. LVESV was unchanged from baseline to 6-month follow-up in the ASC (125.7 ± 68.8 mL and 126.3 ± 72.5 mL, P = 0.827) and placebo (134.6 ± 45.8 mL and 135.3 ± 49.6 mL, P = 0.855) group without any differences between the groups (0.0 mL (95% CI -9.1 to 9.0 mL, P = 0.992). Neither were there significant changes in left ventricular end diastolic volume or LVEF within the two groups or between groups -5.7 mL (95% CI -16.7 to 5.3 mL, P = 0.306) and -1.7% (95% CI -4.4. to 1.0, P = 0.212), respectively). NYHA classification and 6-min walk test did not alter significantly in the two groups (P > 0.05). The quality-of-life, total symptom, and overall summary score improved significantly only in the ASC group but not between groups. There were 24 serious adverse events (SAEs) in the ASC group and 11 SAEs in the placebo group without any significant differences between the two groups at 1-year follow-up. Kaplan-Meier plot using log-rank test of combined cardiac events showed an overall mean time to event of 30 ± 2 months in the ASC group and 29 ± 2 months in the placebo group without any differences between the groups during the 3 years follow-up period (P = 0.994). CONCLUSIONS: Intramyocardial CSCC_ASC injections in patients with chronic HFrEF were safe but did not improve myocardial function or structure, nor clinical symptoms.

Effect of allogeneic adipose tissue-derived mesenchymal stromal cell treatment in chronic ischaemic heart failure with reduced ejection fraction - the SCIENCE trial.

AIMS: The aim of the SCIENCE trial was to investigate whether a single treatment with direct intramyocardial injections of adipose tissue-derived mesenchymal stromal cells (CSCC_ASCs) was safe and improved cardiac function in patients with chronic ischaemic heart failure with reduced ejection fraction (HFrEF). METHODS AND RESULTS: The study was a European multicentre, double-blind, placebo-controlled phase II trial using allogeneic CSCC_ASCs from healthy donors or placebo (2:1 randomization). Main inclusion criteria were New York Heart Association (NYHA) class II-III, left ventricular ejection fraction (LVEF) <45%, and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels >300 pg/ml. CSCC_ASCs or placebo (isotonic saline) were injected directly into viable myocardium. The primary endpoint was change in left ventricular end-systolic volume (LVESV) at 6-month follow-up measured by echocardiography. A total of 133 symptomatic HFrEF patients were included. The treatment was safe without any drug-related severe adverse events or difference in cardiac-related adverse events during a 3-year follow-up period. There were no significant differences between groups during follow-up in LVESV (0.3 ± 5.0 ml, p = 0.945), nor in secondary endpoints of left ventricular end-diastolic volume (-2.0 ± 6.0 ml, p = 0.736) and LVEF (-1.6 ± 1.0%, p = 0.119). The NYHA class improved slightly within the first year in both groups without any difference between groups. There were no changes in 6-min walk test, NT-proBNP, C-reactive protein or quality of life the first year in any groups. CONCLUSION: The SCIENCE trial demonstrated safety of intramyocardial allogeneic CSCC_ASC therapy in patients with chronic HFrEF. However, it was not possible to improve the pre-defined endpoints and induce restoration of cardiac function or clinical symptoms.

Functional in vitro models of the inhibitory effect of adipose tissue-derived stromal cells on lymphocyte proliferation: Improved sensitivity and quantification through flow cytometric analysis.

As the interest in cell-based therapies continue to increase, so does the need for assays detailing potency and providing platforms for identifying mechanisms of action. For most clinical implications of mesenchymal stromal cells, the immunomodulatory effect is crucial. While the suppressive potential on lymphocyte proliferation is well-described in literature, reproducible and standardized assays to document and quantify it varies from research group to research group and between methodologies. The aim of the present study was to utilize flowcytometry to quantify proliferation and identify measurements to increase the assay sensitivity to treatment with adipose tissue-derived stromal cells (ASC). Lymphocyte proliferation was induced by the unspecific mitogen phytohemagglutinin or by alloreactivity towards an irradiated donor in a mixed lymphocyte reaction. Addition of ASC did not change the composition of T cells, B cells, NK cells, NKT cell types considerably; likewise, no increases in proliferation were observed upon inclusion of ASC, demonstrating that ASC does not evoke an additive response. On the contrary, the suppressive effect of ASC was documented. By applying different gating strategies and curve fitting, the sensitivity was increased, and dose-response relationships established. Flow cytometric evaluation allows for more detailed identification of the lymphocytes affected by ASC and constitute a significant asset in future unraveling of modes and mechanisms of action, as well as quantification of potency.

Optimizing an immunomodulatory potency assay for Mesenchymal Stromal Cell.

The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The differences may be attributed to innumerable sources including tissue origin, production methods, or even between batches. While the immunomodulatory potential of MSC is recognized and well-documented by an expansive body of evidence, the methodologies and findings vary markedly. In this study, we utilized flowcytometric analysis of lymphocyte proliferation based on cryopreserved peripheral blood mononuclear cells for quantification of the inhibitory effect of MSC. Technical aspects of fluorescent staining and cryopreservation of peripheral blood mononuclear cells were evaluated to obtain optimal results and increase feasibility. A range of common specific and unspecific mitogens was titrated to identify the conditions, in which the effects of Adipose tissue-derived Stromal Cells (ASC; a type of MSC) were most pronounced. Specific stimulation by antibody-mediated activation of CD3 and CD28 via TransAct and Dynabeads lead to substantial proliferation of lymphocytes, which was inhibited by ASC. These results were closely mirrored when applying unspecific stimulation in form of phytohemagglutinin (PHA), but not concanavalin A or pokeweed mitogen. The mixed lymphocyte reaction is a common assay which exploits alloreactivity between donors. While arguably more physiologic, the output of the assay often varies substantially, and the extent of proliferation is limited since the frequency of alloreactive cells is low, as opposed to the mitogens. To heighten the proliferative response and robustness, combinations of 2-5 donors were tested. Maximum proliferation was observed when combining 4 or more donors, which was efficiently suppressed by ASC. Several desirable and unfavorable traits can be attributed to the tested stimuli in the form of keywords. The importance of these traits should be scored on a laboratory-level to identify the ideal mitogen. In our case the ranking listed PHA as the most suited candidate. Developing robust assays is no trivial feat. By disclosing the full methodological framework in the present study, we hope to aid others in establishing functional metrics on the road to potency assays.

Intraglandular mesenchymal stem cell treatment induces changes in the salivary proteome of irradiated patients.

BACKGROUND: Hyposalivation and xerostomia (dry mouth), are the leading site-effects to treatment of head and neck cancer. Currently, there are no effective therapies to alleviate radiation-induced hyposalivation. Adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) have shown potential for restoring salivary gland function. However, the mode of action is unknown. The purpose of the present study was therefore to characterize the effect of AT-MSC therapy on the salivary proteome in previously irradiated head and neck cancer patients. METHODS: Whole saliva was collected from patients with radiation-induced salivary gland hypofunction (n = 8) at baseline, and 120 days after AT-MSC treatment, and from healthy controls (n = 10). The salivary proteome was characterized with mass spectrometry based proteomics, and data was compared within the AT-MSC group (baseline versus day 120) and between AT-MSC group and healthy controls. Significance levels between groups were determined by using double-sided t-test, and visualized by means of principal component analysis, volcano plots and cluster analysis. RESULTS: Here we show that 140 human proteins are significantly differentially expressed in saliva from patients with radiation-induced hypofunction versus healthy controls. AT-MSC treatment induce a significant impact on the salivary proteome, as 99 proteins are differentially expressed at baseline vs. 120 days after treatment. However, AT-MSC treatment does not restore healthy conditions, as 212 proteins are significantly differentially expressed in saliva 120 days after AT-MSCs treatment, as compared to healthy controls. CONCLUSION: The results indicate an increase in proteins related to tissue regeneration in AT-MSCs treated patients. Our study demonstrates the impact of AT-MSCs on the salivary proteome, thereby providing insight into the potential mode of action of this novel treatment approach.

Currently, there are no effective treatments to ease dry mouth, which is a leading long-term side effect of radiation treatment for head and neck cancer. However, treatment with stem cells has shown potential for restoring function of the salivary glands, which are damaged due to radiation. We compared proteins in saliva of previously radiation-treated patients with healthy non-irradiated persons and found differences in the levels of 140 proteins. After stem cell treatment of irradiated patients, we found changes in the salivary content of proteins related to tissue regeneration. Our study demonstrates the impact of stem cell treatment on proteins in saliva, thereby providing insight into the potential mode of action of this treatment approach for patients with radiation-induced dry mouth. Consequently, this could potentially help to improve treatment of dry mouth in the future.

Evaluation of the α-synuclein PET radiotracer (d3)-[11C]MODAG-001 in pigs.

BACKGROUND: A positron emission tomography (PET) radiotracer to neuroimage α-synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinson's disease, where elevated aggregate levels are a histopathological hallmark. The radiotracer (d3)-[11C]MODAG-001 has recently shown promise for visualization of α-synuclein pre-formed fibrils (α-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 µg α-PFF, and in the other hemisphere, either 75 µg α-PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimer's disease (AD) was injected. All pigs underwent one or two (d3)-[11C]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region. RESULTS: The α-PFF and AD homogenate injected brain regions had high uptake of (d3)-[11C]MODAG-001 compared to the occipital cortex or cerebellum. BPND values in 150 µg α-PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BPND values in the 150 µg and 75 µg injected regions of α-PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001. CONCLUSION: We find that both α-PFF and AD brain homogenates give rise to increased binding of (d3)-[11C]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral α-synuclein pathology, (d3)-[11C]MODAG-001 shows promise as a lead tracer for future radiotracer development.

Adipose-derived stromal cells increase the formation of collagens through paracrine and juxtacrine mechanisms in a fibroblast co-culture model utilizing macromolecular crowding.

BACKGROUND: Adipose-derived stromal cells (ASCs) possess a multitude of regenerative capabilities, which include immunomodulation, angiogenesis, and stimulation of extracellular matrix (ECM) remodeling. However, the underlying mechanisms leading to ECM remodeling remain largely elusive and highlight the need for functional in vitro models for mode of action studies. Therefore, the purpose of this study was to develop an in vitro co-culture model to investigate the capabilities of ASCs to modulate fibroblasts and ECM. METHODS: An ECM in vitro model with ASCs and normal human dermal fibroblasts (NHDFs) was established utilizing macromolecular crowding, ascorbic acid, and TGF-ß stimulation. Paracrine and juxtacrine co-cultures were created using transwell inserts and cell cultures with direct cell-cell contacts. The cultures were screened using RT2 PCR Profiler Arrays; the protein levels of myofibroblast differentiation marker alpha smooth muscle actin (αSMA) and ECM remodeling enzymes were analyzed using western blot on cell lysates; the formation of collagen type I, III, VI, and fibronectin was investigated using ELISA on culture supernatants; and the deposition of collagens was analyzed using immunocytochemistry. RESULTS: TGF-ß stimulation of NHDF monocultures increased the expression of 18 transcripts relevant for ECM formation and remodeling, the protein levels of αSMA and matrix metalloproteinase-2 (MMP-2), the formation of collagen type I, III, VI, and fibronectin, and the deposition of collagen type I and VI and decreased the protein levels of MMP-14. Inclusion of ASCs in the ECM co-culture model increased the formation of collagen type I and III through paracrine mechanisms and the formation of collagen type VI through juxtacrine mechanisms. CONCLUSIONS: The co-culture model provides effective stimulation of NHDF monocultures by TGF-ß for enhanced formation and deposition of ECM. In the model, ASCs induce changes in ECM by increasing formation of collagen type I, III and VI. The obtained results could guide further investigations of ASCs' capabilities and underlying mechanisms related to ECM formation and remodeling.

An in vivo Pig Model for Testing Novel Positron Emission Tomography Radioligands Targeting Cerebral Protein Aggregates.

Positron emission tomography (PET) has become an essential clinical tool for diagnosing neurodegenerative diseases with abnormal accumulation of proteins like amyloid-ß or tau. Despite many attempts, it has not been possible to develop an appropriate radioligand for imaging aggregated α-synuclein in the brain for diagnosing, e.g., Parkinson's Disease. Access to a large animal model with α-synuclein pathology would critically enable a more translationally appropriate evaluation of novel radioligands. We here establish a pig model with cerebral injections of α-synuclein preformed fibrils or brain homogenate from postmortem human brain tissue from individuals with Alzheimer's disease (AD) or dementia with Lewy body (DLB) into the pig's brain, using minimally invasive surgery and validated against saline injections. In the absence of a suitable α-synuclein radioligand, we validated the model with the unselective amyloid-ß tracer [11C]PIB, which has a high affinity for ß-sheet structures in aggregates. Gadolinium-enhanced MRI confirmed that the blood-brain barrier was intact. A few hours post-injection, pigs were PET scanned with [11C]PIB. Quantification was done with Logan invasive graphical analysis and simplified reference tissue model 2 using the occipital cortex as a reference region. After the scan, we retrieved the brains to confirm successful injection using autoradiography and immunohistochemistry. We found four times higher [11C]PIB uptake in AD-homogenate-injected regions and two times higher uptake in regions injected with α-synuclein-preformed-fibrils compared to saline. The [11C]PIB uptake was the same in non-injected (occipital cortex, cerebellum) and injected (DLB-homogenate, saline) regions. With its large brain and ability to undergo repeated PET scans as well as neurosurgical procedures, the pig provides a robust, cost-effective, and good translational model for assessment of novel radioligands including, but not limited to, proteinopathies.

Cryopreservation of peripheral blood mononuclear cells for use in proliferation assays: First step towards potency assays.

Investigational cell-based therapeutics are rapidly heading towards pivotal clinical trials. The premise is that the scientific rationale is well defined, and that product quality reflects exactly this. In vitro potency assays are necessary tools for evaluating cell products, and with potency assays comes high demands for standardization and reproducibility of the methods involved. For demonstrating principles of cell therapeutics for allogeneic use or with claimed immunosuppressive efficacies, assays involving peripheral blood mononuclear cells (PBMC) are critical. Establishment of a cryopreserved bank of PBMC favors standardization, as it allows repeated use of a single donor and simultaneous testing of several donors. The first step to fulfil such potential is to ensure optimum conditions for preservation of PBMC function, and secondly to design assays which heightens the reproducibility. Emphasis should be put on application of the assay. The objective of the present study was to establish a methodological foundation for cell therapeutics to be tested, and several aspects were factored in, including cell concentrations and partial changes of medium. PBMC were isolated and cryopreserved in six formulations of cryoprotective medium consisting of fetal bovine serum (90%, 60%, and 30%) in combination with dimethyl sulfoxide (10% or 5%). The proliferative capacity of the cryopreserved cells was assayed by labeling with carboxyfluorescein succinimidyl ester and stimulation by phytohemagglutinin or in mixed lymphocyte reactions, analyzed by flow cytometry. To counter an eventual lag phase post thaw, the assays were designed to include two durations and to explore the possibility of reducing cell numbers, two cell concentrations. Qualitative and quantitative aspects of the staining were affected by formulation as well as design, stressing the importance of basic optimization for assay development. We conclude that the established methods allow for optimized preservation of function and will serve as a platform for further development of robust functional assays.