Explant Research Articles

In vitro myelination using explant culture of dorsal root ganglia: An efficient tool for analyzing peripheral nerve differentiation and disease modeling.

Peripheral nerves conducting motor and somatosensory signals in vertebrate consist of myelinated and unmyelinated axons. In vitro myelination culture, generated by co-culturing Schwann cells (SCs) and dorsal root ganglion (DRG) neurons, is an indispensable tool for modeling physiological and pathological conditions of the peripheral nervous system (PNS). This technique allows researchers to overexpress or downregulate molecules investigated in neurons or SCs to evaluate the effect of such molecules on myelination. In vitro myelination experiments are usually time-consuming and labor-intensive to perform. Here we report an optimized protocol for in vitro myelination using DRG explant culture. We found that our in vitro myelination using DRG explant (IVMDE) culture not only achieves myelination with higher efficiency than conventional in vitro myelination methods, but also can be used to observe Remak bundle and non-myelinating SCs, which were unrecognizable in conventional methods. Because of these characteristics, IVMDE may be useful in modeling PNS diseases, including Charcot Marie Tooth disease (CMT), in vitro. These results suggest that IVMDE may achieve a condition more similar to peripheral nerve myelination observed during physiological development.

Evaluating the effect of gamma rays on Zamiifolia (Zamioculcas zamiifolia) plant in vitro and genetic diversity of the resulting genotypes using the ISSR marker

Zamiifolia (Zamioculcas sp.) is a perennial plant in the Araceae family and one of the new apartment plants in the world. In this study, in order to increase the effectiveness of the breeding program, tissue culture technique and explants of leaf parts were used. The results indicated that 2,4-D (1 mg/l) and BA (2 mg/l) hormones affected positively and significantly callus formation and simultaneous application of NAA and BA (both in 0.5 mg/l) caused the best results regarding seedling production and number, leaves, complete tubers, and root in tissue culture of Zaamifolia. In the study, three cultivars of Zamiifolia (green, black and Dutch) and 12 genotypes resulted from callus formation stage, irradiated with different gamma rays (0 to 175 Gy, with LD50 as 68 Gy) were selected and the presence of genetic diversity was investigated using 22 ISSR primers. Applying ISSR marker showed that the highest amount of PIC values related to the F19(0.47) and F20(0.38) primers, which persuasively isolated the studied genotypes. Moreover, the highest efficiency was detected for AK66 marker based on the MI parameter. The PCA and clustering categorization via UPGMA methodology based on molecular information and Dice index, differentiated the genotypes into 6 groups. Genotypes 1(callus), 2(100 Gy) and 3(cultivar from Holland) created separated groups. The 4th group included 6(callus), 8(0 Gy), 9(75 Gy), 11(90 Gy), 12(100 Gy) and 13(120 Gy) genotypes appearing as the largest group. The 5th group included 7(160 Gy), 10(80 Gy), 14(140 Gy) and 15(Zanziber gem black) genotypes. The last group included 4(mather plant) and 5(callus) genotypes. In this context, genotypes 1, 5, and 6 had probably somaclonal variation. Moreover, genotypes that received doses of 100 and 120 Gy, had a medium diversity. There is a high possibility of introducing a cultivar with a low dose and high genetic diversity in the whole group. Genotype 7 in this classification, received the highest dose of 160 Gy. In this population, the Dutch variety, was used as a new variety. As a result, the ISSR marker could correctly group the genotypes. This is an interesting finding, and it could be hypothesized that the ISSR marker could correctly differentiate Zaamifolia genotypes and probably other ornamental plants under the effect of gamma rays mutagenesis in order to achieve novel variants.

Isolation methods, proliferation, and adipogenic differentiation of adipose-derived stem cells from different fat depots in bovines.

The adipose-derived stem cells (ASCs) are a valuable resource for regenerative medicine and essential materials for research in fat deposition. However, the isolation procedure of ASCs has not been standardized and needs to be harmonized; differences in proliferation and adipogenic differentiation of ASCs obtained from different fat depots have not been well characterized. In the present study, we compared the efficiency of ASCs isolation by enzymatic treatment and explant culture methods and the proliferation ability and adipogenic differentiation potential of ASCs isolated from subcutaneous and visceral fat depots. The explant culture method was simple and with no need for expensive enzymes while the enzymatic treatment method was complex, time consuming and costly. By the explant culture method, a larger number of ASCs were isolated from subcutaneous and visceral fat depots. By contrast, fewer ASCs were obtained by the enzymatic treatment method, especially from visceral adipose. ASCs isolated by the explant culture method performed well in cell proliferation and adipogenic differentiation, though they were slightly lower than those by the enzymatic treatment method. ASCs isolated from visceral depot demonstrated higher proliferation ability and adipogenic differentiation potential. In total, the explant culture method is simpler, more efficient, and lower cost than the enzymatic treatment method for ASCs isolation; compared with visceral adipose, subcutaneous adipose is easier to isolate ASCs; however, the visceral ASCs are superior to subcutaneous ASCs in proliferation and adipogenic differentiation.

Maternal elevated inflammation impairs placental fatty acids β-oxidation in women with gestational diabetes mellitus.

An adverse proinflammatory milieu contributes to abnormal cellular energy metabolism response. Gestational diabetes mellitus (GDM) is closely related to an altered maternal inflammatory status. However, its role on lipid metabolism regulation in human placenta has not yet been assessed. The aim of this study was to examine the impact of maternal circulating inflammatory mediators ([TNF]-α, [IL]-6, and Leptin) on placental fatty acid metabolism in GDM pregnancies. Fasting maternal blood and placental tissues were collected at term deliveries from 37 pregnant women (17 control and 20 GDM). Molecular approach techniques as radiolabeled lipid tracers, ELISAs, immunohistochemistry and multianalyte immunoassay quantitative analysis, were used to quantify serum inflammatory factors' levels, to measure lipid metabolic parameters in placental villous samples (mitochondrial fatty acid oxidation [FAO] rate and lipid content [Triglycerides]), and to analyze their possible relationships. The effect of potential candidate cytokines on fatty acid metabolism in ex vivo placental explants culture following C-section a term was also examined. Maternal serum IL-6, TNF-α and leptin levels were significantly increased in GDM patients compared with control pregnant women (9,9±4,5 vs. 3,00±1,7; 4,5±2,8 vs. 2,1±1,3; and 10026,7±5628,8 vs. 5360,2±2499,9 pg/ml, respectively). Placental FAO capacity was significantly diminished (~30%; p<0.01), whereas triglyceride levels were three-fold higher (p<0.01) in full-term GDM placentas. Uniquely the maternal IL-6 levels showed an inverse and positive correlation with the ability to oxidize fatty acids and triglyceride amount in placenta, respectively (r= -0,602, p=0.005; r= 0,707, p=0.001). Additionally, an inverse correlation between placental FAO and triglycerides was also found (r=-0.683; p=0.001). Interestingly, we ex vivo demonstrated by using placental explant cultures that a prolonged exposure with IL-6 (10 ng/mL) resulted in a decline in the fatty acid oxidation rate (~25%; p=0.001), along to acute increase (2-fold times) in triglycerides accumulation (p=0.001), and in lipid neutral and lipid droplets deposits. Enhanced maternal proinflammatory cytokines levels (essentially IL-6) is closely associated with an altered placental fatty acid metabolism in pregnancies with GDM, which may interfere with adequate delivery of maternal fat across the placenta to the fetus.

Primary Culture of Dental Pulp Stem Cells.

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

Matrix metalloproteinases: expression and regulation in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs.

Matrix metalloproteinases (MMPs) are a family of endoproteases produced by various tissues and cells and play important roles in angiogenesis, tissue repair, immune response, and endometrial remodeling. However, the expression and function of MMPs in the pig endometrium during the estrous cycle and pregnancy have not been fully elucidated. Thus, we determined the expression, localization, and regulation of MMP2, MMP8, MMP9, MMP12, and MMP13 in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs. Endometrial tissues during the estrous cycle and pregnancy and conceptus and chorioallantoic tissues during pregnancy were obtained and the expression of MMPs was analyzed. The effects of steroid hormones and cytokines on the expression of MMPs were determined in endometrial explant cultures. Expression levels of MMP12 and MMP13 changed during the estrous cycle, while expression of MMP2, MMP9, MMP12, and MMP13 changed during pregnancy. Expression of MMP2, MMP8, and MMP13 mRNAs was cell type-specific at the maternal-conceptus interface. Gelatin zymography showed that enzymatically active MMP2 was present in endometrial tissues. In endometrial explant cultures, estradiol-17β induced the expression of MMP8 and MMP12, progesterone decreased the expression of MMP12, interleukin-1β increased the expression of MMP2, MMP8, MMP9, and MMP13, and interferon-γ increased the expression of MMP2. These results suggest that MMPs expressed in response to steroids and cytokines play an important role in the establishment and maintenance of pregnancy by regulating endometrial remodeling and processing bioactive molecules in pigs.

A Novel Tissue Explant Culture Model For Testing of Candidate Treatments for Inflammation of the Uterus in Pigs.

Abstract Background: Endometritis is an inflammatory condition of the uterus leading to complications with reproductive organs, miscarriage and infertility. The first-line treatment of endometritis in pigs are antibiotics, which do not always resolve inflammation and contribute to antibiotic resistance. There is an urgent need for the development of alternative treatments for endometritis. Here we propose and validate the first porcine tissue explant model for uterine inflammation and test naturally derived compounds for their effectiveness as anti-inflammatory therapies in porcine endometrial tissue. Methods: Uterine endometrial explants were harvested from uteri of sows sent to slaughter. Tissue biopsies were collected at follicular and luteal phases of the oestrous cycle. Biopsies were washed and these tissue ‘explants’ were cultured. Explants were treated at 24 hr with oxytocin (to stimulate PGF 2αproduction) or LPS (to mimic infection and induce inflammation. Explants were retained for RT-PCR analysis. Supernatants were harvested and concentrations of markers were measured by ELISA and Luminex. Results: The data generated thus far show a dose dependent response of IL-6 secretion to LPS stimulation, which demonstrates the validity of our model. Inflammation can be stimulated in an in vitrosetting to mimic porcine endometritis and we present data to show the change in cytokine levels in response to treatment with naturally derived compounds. Conclusions: This data shows that inflammation can be stimulated in an in vitrosetting to mimic porcine endometritis This model is a tool for testing of novel compounds and their potential anti-inflammatory effects on the porcine endometrium. Supported by an Innovate UK Grant (Project Reference: 98015) (https://gtr.ukri.org/projects?ref=98015)

An animal model for autoinflammation with infantile enterocolitis

Abstract Autoinflammation with infantile enterocolitis (AIFEC) is one of the emerging life-threatening diseases caused by inborn errors of NLRC4 inflammasome. The pathogenicity of AIFEC remains largely unknown and standard therapy is lacking due to rare patient cases and high mortality. To fill the gap, it is urgent to set up an effective animal model for this rare disease. We have recently generated a loxP-flanked NLRC4 V341A conditional knock-in (KI) mouse line where human V341A (GTG to GCG) mutation was introduced into the corresponding mouse Nlrc4coding sequence. When this line is bred with distinct Cre transgenic mice, the KI allele can be expressed globally or cell-type specifically. We bred the KI line with E2a-Cre transgenic mice and got globally expressed V341A KI mice, and the KI mice developed autoinflammation with infantile enterocolitis that recapitulated the symptoms of AIFEC patients. In brief, immunoblot demonstrated KI mice had hyperactivation of NLRC4 inflammasome in gut epithelium and macrophages. The neonatal KI pups had significant intestinal inflammation, accompanied with diarrheal, impaired barrier integrity and dysbiosis. Furthermore, the KI pups had dramatically elevated IL-1β, IL-6 and IL-18 in sera and supernatant of colon explant cultures. Meanwhile, the KI pups exhibited cytopenia, hemophagocytosis, and macrophage hyperactivation. Biochemical assays suggested multi-organ damage in KI mice, including the liver, kidney, and heart, accompanied with hyper serum ferritin level. Without any intervention, the pups died before day 10 postnatally. Taken together, we have established a novel animal model for AIFEC, thus providing a platform to elucidate the pathogenesis of AIFEC and test therapeutic strategies. Startup fund from the Department of pathology at the University of IOWA, NIH R21AG076895, R01 NS104164

Mincle-GSDMD-mediated release of IL-1β small extracellular vesicles from hepatic macrophages in ethanol-induced liver injury.

Macrophage-inducible C-type lectin (Mincle) is expressed on hepatic macrophages and senses ethanol (EtOH)-induced danger signals released from dying hepatocytes and promotes IL-1β production. However, it remains unclear what and how EtOH-induced Mincle ligands activate downstream signaling events to mediate IL-1β release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating β-glucosylceramide (β-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which β-GluCer engages Mincle on hepatic macrophages to release IL-1β in the absence of cell death and exacerbates ALD. Concentrations of β-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of hepatic macrophages with lipopolysaccharide and β-GluCer induced formation of a Mincle and Gsdmd-dependent secretory complex containing chaperoned full-length gasdermin D (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1β and components of the Caspase 8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEVs). Gao-binge EtOH exposure to wild-type, but not Mincle-/- and Gsdmd-/-, mice increased release of IL-1β-containing sEVs from liver explant cultures. Myeloid-specific deletion of Gsdmd similarly decreased the formation of sEVs by liver explant cultures and protected mice from EtOH-induced liver injury. sEVs collected from EtOH-fed wild-type, but not Gsdmd-/-, mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge EtOH-induced liver injury. β-GluCer functions as a danger-associated molecular pattern activating Mincle-dependent gasdermin D-mediated formation and release of IL-1β-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD.

Induction of Axillary Bud Swelling of Hevea brasiliensis to Regenerate Plants through Somatic Embryogenesis and Analysis of Genetic Stability.

To overcome rubber tree (RT) tissue culture explant source limitations, the current study aimed to establish a new Hevea brasiliensis somatic embryogenesis (SE) system, laying the technical foundation for the establishment of an axillary-bud-based seedling regeneration system. In this study, in vitro plantlets of Hevea brasiliensis Chinese Academy of Tropical Agricultural Sciences 917 (CATAS 917) were used as the experimental materials. Firstly, the optimum conditions for axillary bud swelling were studied; then, the effects of phenology, the swelling time of axillary buds (ABs), and medium of embryogenic callus induction were studied. Plantlets were obtained through somatic embryogenesis. Flow cytometry, inter-simple sequence repeat (ISSR molecular marker) and chromosome karyotype analysis were used to study the genetic stability of regenerated plants along with budding seedlings (BSs) and secondary somatic embryo seedlings (SSESs) as the control. The results show that the rubber tree's phenology period was mature, and the axillary bud induction rate was the highest in the 2 mg/L 6-benzyladenine (6-BA) medium (up to 85.83%). Later, 3-day-old swelling axillary buds were used as explants for callogenesis and somatic embryogenesis. The callus induction rate was optimum in MH (Medium in Hevea) + 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) + 1.5 mg/L 1-naphthalene acetic acid (NAA) + 1.5 mg/L Kinetin (KT) + 70 g/L sucrose (56.55%). The regenerated plants were obtained after the 175-day culture of explants through callus induction, embryogenic callus induction, somatic embryo development, and plant regeneration. Compared with the secondary somatic embryo seedling control, axillary bud regeneration plants (ABRPs) were normal diploid plants at the cellular and molecular level, with a variation rate of 7.74%.

Oleic acid abomasal infusion limits lipolysis and improves insulin sensitivity in adipose tissue from periparturient dairy cows

Excessive adipose tissue (AT) lipolysis around parturition in dairy cows is associated with impaired AT insulin sensitivity and increased incidence of metabolic diseases. Supplementing cows with oleic acid (OA) reduces circulating biomarkers of lipolysis and improves energy balance. Nevertheless, it is unclear if OA alters lipid trafficking in AT. In the liver and skeletal muscle, OA improves mitochondrial function and promotes lipid droplet formation by activating perilipin 5 (PLIN5) and peroxisome proliferator-activated receptor α (PPARα). However, it is unknown if this mechanism occurs in AT. The objective of this study was to determine the effect of OA on AT lipolysis, systemic and AT insulin sensitivity, and AT mitochondrial function in periparturient dairy cows. Twelve rumen-cannulated Holstein cows were infused abomasally following parturition with ethanol (CON) or OA (60 g/d) for 14 d. Subcutaneous AT samples were collected at 11 ± 3.6 d before calving (-12 d), and 6 ± 1.0 d (7 d) and 13 ± 1.4 d (14 d) after parturition. An intravenous glucose tolerance test was performed on d 14. Adipocyte morphometry was performed on hematoxylin and eosin-stained AT sections. The antilipolytic effect of insulin (1 μg/L) was evaluated using an ex vivo explant culture following lipolysis stimulation. PLIN5 and PPARα transcription and translation were determined by real-time quantitative PCR and capillary electrophoresis, respectively. RNA sequencing was used to evaluate the transcriptomic profile of mitochondrial gene networks. In CON cows, postpartum lipolysis increased the percentage of smaller (<3,000 µm2) adipocytes at 14 d compared with -12 d. However, OA limited adipocyte size reduction at 14 d. Likewise, OA decreased lipolysis plasma markers nonesterified free fatty acids and β-hydroxybutyrate at 5 and 7 d. Over the 14-d period, compared with CON, OA increased the concentration of plasma insulin and decreased plasma glucose. During the glucose tolerance test, OA decreased circulating glucose concentration (at 10, 20, 30, 40 min) and the glucose clearance rate. Moreover, OA increased insulin at 10 and 20 min and tended to increase it at 30 min. Following lipolysis stimulation, OA improved the antilipolytic effect of insulin in the AT at 14 d. PLIN5 and PPARA gene expression decreased postpartum regardless of treatment. However, OA increased PLIN5 protein expression at 14 d and increased PPARA at 7 and 14 d. Immunohistochemical analysis of AT and RNA sequencing data showed that OA increased the number of mitochondria and improved mitochondrial function. However, OA had no effect on production and digestibility. Our results demonstrate that OA limits AT lipolysis, improves systemic and AT insulin sensitivity, and is associated with markers of mitochondrial function supporting a shift to lipogenesis in AT of periparturient dairy cows.

P158 Defining distinct fibroblast populations in systemic sclerosis skin using functional characterization and single-cell RNA sequencing

Abstract Background/Aims Fibroblasts isolated by traditional explant culture from skin biopsies in systemic sclerosis (SSc) have a profibrotic and “migratory” phenotype. Single cell RNAseq (scRNAseq) studies suggest other fibroblast subpopulations may be important in SSc pathogenesis. We have isolated a second fibroblast population termed “resident” from whole skin in SSc. We used bulk and scRNAseq to further characterise these populations. Methods Forearm skin from dcSSc (n = 3) and HC (n = 3) was biopsied. “Migratory” fibroblasts were isolated by standard explant culture. The remaining biopsy fragments underwent collagenase digestion to yield a second “resident” fibroblast population. Functional characterization included 3-D collagen gel contraction, migratory scratch-wound assays and western blot comparing expression of pro-collagen I, CTGF and αSMA. Significant difference in gene expression was defined by fold change≥1.5, and adjusted p-value&amp;lt;0.05. scRNAseq was performed on 12 SSc whole skin biopsies, and 3 HC, using the 10X Genomics platform. Results Both SSc fibroblast populations showed a fibrotic phenotype with overexpression of COL1, CTGF and αSMA compared to HC, but with reduced expression αSMA in resident compared to migratory SSc fibroblasts (p &amp;lt; 0.05). RNAseq confirmed 1483 genes being significantly differentially expressed between the SSc fibroblast populations (Table 1). All four populations clustered separately on hierarchical clustering. 10 fibroblast clusters were identified using scRNAseq of whole skin. Of these, cluster 0 and 4 showed greater expression of COL10A1, and STC2 in keeping with migratory fibroblasts. Increased expression of CCL2, and EGR1 were seen in clusters 3 and 6, representing the resident fibroblast population. Gene set enrichment analysis (GSEA) confirmed concordance between KEGG pathway upregulation of the migratory and resident fibroblast clusters and bulk RNAseq analysis. Resident fibroblasts showed upregulation of complement and coagulation genes, and JAK/STAT signalling pathway. There was upregulation of glycolysis gluconeogenesis, and PPAR signalling in migratory fibroblasts. There was agreement in GSEA findings between relevant scRNAseq clusters and bulkRNAseq for resident and migratory subpopulations. Conclusion We have isolated for the first time two distinct profibrotic fibroblast populations from SSc skin with altered expression of key genes associated with SSc pathogenesis. Future work will define their role in SSc pathogenesis, potentially facilitating a more targeted treatment approach. Disclosure K.E. Clark: None. S. Xu: None. V.H. Ong: None. C.D. Buckley: None. C. Denton: None.

Pyruvate-conjugation of PEGylated liposomes for targeted drug delivery to retinal photoreceptors

Despite several promising candidates, there is a paucity of drug treatments available for patients suffering from retinal diseases. An important reason for this is the lack of suitable delivery systems that can achieve sufficiently high drug uptake in the retina and its photoreceptors. A promising and versatile method for drug delivery to specific cell types involves transporter-targeted liposomes, i.e., liposomes surface-coated with substrates for transporter proteins highly expressed on the target cell. We identified strong lactate transporter (monocarboxylate transporter, MCT) expression on photoreceptors as a potential target for drug delivery vehicles. To evaluate MCT suitability for drug targeting, we used PEG-coated liposomes and conjugated these with different monocarboxylates, including lactate, pyruvate, and cysteine. Monocarboxylate-conjugated and dye-loaded liposomes were tested on both human-derived cell-lines and murine retinal explant cultures. We found that liposomes conjugated with pyruvate consistently displayed higher cell uptake than unconjugated liposomes or liposomes conjugated with lactate or cysteine. Pharmacological inhibition of MCT1 and MCT2 reduced internalization, suggesting an MCT-dependent uptake mechanism. Notably, pyruvate-conjugated liposomes loaded with the drug candidate CN04 reduced photoreceptor cell death in the murine rd1 retinal degeneration model while free drug solutions could not achieve the same therapeutic effect. Our study thus highlights pyruvate-conjugated liposomes as a promising system for drug delivery to retinal photoreceptors, as well as other neuronal cell types displaying high expression of MCT-type proteins.

Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications

BackgroundMesenchymal stem cells (MSCs) have been proven to prevent and clear corneal scarring and limbal stem cell deficiency. However, using animal-derived serum in a culture medium raises the ethical and regulatory bar. This study aims to expand and characterize human limbus-derived stromal/mesenchymal stem cells (hLMSCs) for the first time in vitro in the xeno-free medium.MethodsLimbal tissue was obtained from therapeutic grade corneoscleral rims and subjected to explant culture till tertiary passage in media with and without serum (STEM MACS XF; SM), to obtain pure hLMSCs. Population doubling time, cell proliferation, expression of phenotypic markers, tri-lineage differentiation, colony-forming potential and gene expression analysis were carried out to assess the retention of phenotypic and genotypic characteristics of hLMSCs.ResultsThe serum-free medium supported the growth of hLMSCs, retaining similar morphology but a significantly lower doubling time of 23 h (*p < 0.01) compared to the control medium. FACS analysis demonstrated ≥ 90% hLMSCs were positive for CD90+, CD73+, CD105+, and ≤ 6% were positive for CD45−, CD34−andHLA-DR−. Immunofluorescence analysis confirmed similar expression of Pax6+, COLIV+, ABCG2+, ABCB5+, VIM+, CD90+, CD105+, CD73+, HLA-DR−andCD45−, αSMA− in both the media. Tri-lineage differentiation potential and gene expression of hLMSCs were retained similarly to that of the control medium.ConclusionThe findings of this study demonstrate successful isolation, characterization and culture optimization of hLMSCs for the first time in vitro in a serum-free environment. This will help in the future pre-clinical and clinical applications of MSCs in translational research.

Prenatal triclosan exposure impairs mammalian lung branching morphogenesis through activating Bmp4 signaling

Triclosan (TCS) is a commonly used antibacterial agent present in personal care and household products. Recently, there have been increasing concerns about the association between children’s health and TCS exposure during gestation, but the toxicological effects of TCS exposure on embryonic lung development remain undetermined. In this study, through using an ex vivo lung explant culture system, we found that prenatal exposure to TCS resulted in impaired lung branching morphogenesis and altered proximal-distal airway patterning. These TCS-induced dysplasias are accompanied by significantly reduced proliferation and increased apoptosis within the developing lung, as a consequence of activated Bmp4 signaling. Inhibition of Bmp4 signaling by Noggin partially rescues the lung branching morphogenesis and cellular defects in TCS-exposed lung explants. In addition, we provided in vivo evidence that administration of TCS during gestation leads to compromised branching formation and enlarged airspace in the lung of offspring. Thus, this study provides novel toxicological information on TCS and indicated a strong/possible association between TCS exposure during pregnancy and lung dysplasia in offspring.

Abstract 4122: TIL-containing patient-derived explant cultures reveal role of metformin on antigen presenting cell activation

Abstract Globally, breast cancer is among the most diagnosed cancer types for women. Current and upcoming breast cancer therapies are being investigated in combination with compounds that stimulate an immune response, but whether the therapeutic agents themselves have unexpected immunomodulatory effects is often overlooked. Here, we have developed a method to grow 3D cultures of intact fragments of patient-derived tissue (Patient-Derived Explant Cultures; PDECs) to assess the preclinical potential of studying human tumor cells and immune cells simultaneously ex vivo Single cell sequencing, flow cytometry, gene expression profiling and cytokine profiling data show that the tumor immunocontexture is conserved in PDECs and that these resident immune cells respond to distinct immune stimulus We performed gene expression profiling, flow cytometry, and cytokine profiling of drug-treated human explants and found that metformin has antitumor potential through the activation of antigen presenting cells. We further validated in vitro that metformin-mediated APC activation is largely through mitochondrial respiration inhibition irrespective of the presence of tumor cells. Our PDEC platform highlights the preclinical potential of ex vivo explants by simultaneously offering information of tumor and immune cell toxicity and mechanism. Citation Format: Rita J. Turpin, Ruixian Liu, Pauliina Munne, Aino Peura, Jenna Rannikko, Gino Philips, Natasha Salmelin, Elina Hurskainen, Ilida Suleymanova, Minna Mutka, Tuomo Meretoja, Johanna Mattson, Satu Mustjoki, Päivi Saavalainen, Diether Lambrechts, Jeroen Pouwels, Maija Hollmén, Juha Klefström. TIL-containing patient-derived explant cultures reveal role of metformin on antigen presenting cell activation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4122.

Abstract 1725: Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma

Abstract Approximately 95% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, accounting for over 45,000 deaths per year in the US. In KRAS-mutant cells, KRAS protein exists predominantly in an active, GTP-bound state, leading to excessive oncogenic signaling via RAF/MEK/ERK and other effector pathways. After decades of anticipation, the first therapeutic inhibitors of this oncoprotein target a specific mutant allele (KRASG12C) and have shown promising results in this rare subset of human PDAC patients (1-2% of cases). While these encouraging clinical results bolster support for targeting RAS in human PDAC, most patients harbor KRASG12D and KRASG12V mutations and remain unserved by targeted therapies. Here we report preclinical studies of RM-042, a first-in-class, potent, oral, small molecule inhibitor of GTP-bound RAS proteins, designed to treat cancers driven by a variety of RAS mutations. RMC-6236, a clinical candidate and RM-042, a tool compound, both act through non-covalent binding to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages GTP-bound RAS to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors.We examined the anti-tumor activity of RM-042 in two complementary preclinical model systems of PDAC. Using primary human PDAC explant cultures, we found that RM-042 potently inhibits phospho-ERK expression (a marker of KRAS/RAF/MEK pathway activity), in a concentration-dependent manner and was associated with reduced viability and altered stromal content. Pilot short-term studies in the K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) mouse model of PDAC found that RM-042 induced initial tumor stabilizations and regressions associated with loss of pERK expression, in association with increased apoptosis and reduced proliferation, at tolerated doses. However, tumors collected at endpoints showed re-established pERK expression, suggesting activation of resistance/adaptation mechanisms. Ongoing studies will determine pharmacokinetic properties of RM-042 in the autochthonous PDAC mouse model, as well as the duration of tumor responses and evaluate a potential survival benefit provided by RM-042. Regulatory network analysis of single cell RNA sequencing performed on these tumors at multiple timepoints will provide a mechanistic understanding of the regulatory proteins that drive responses in each cell type in the tumors. Through this approach, we will assess the regulatory adaptations that occur as tumors initially respond and as they escape. Together, these studies provide complementary evidence from in vivo murine and ex vivo human models for promising anti-tumor activity in response to GTP-RAS inhibition, supporting the inclusion of PDAC patients in ongoing and future clinical trials of RMC-6236. Citation Format: Urszula N. Wasko, Stephen A. Sastra, Carmine F. Palermo, David Wildes, Mallika Singh, Kenneth P. Olive. Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1725.

Abstract 4910: Human tumor primary culture analyses for the prediction of response to chemotherapy, targeted agents, drug combinations and metabolic inhibitors: The role of ex vivo analysis of programmed cell death in cancer therapy

Abstract There is growing interest in the use of human tumor primary cultures for the prediction of response to drugs and drug combinations in cancer medicine. While the field has witnessed numerous attempts to apply human tissue for the prediction of response, fundamental errors in methodologic approaches severely limited the predictive validity and clinical applicability of these important technologies. The first error was a focus upon drug induced cell growth inhibition. This lead to the failure of the clonogenic (human tumor stem cell) and H3*Thymidine incorporation platforms in the 1970’s and 1980’s. As the concept of apoptosis arose following landmark observations in 1972, cell death endpoints became predominant including mitochondrial function (MTT, XTT), ATP content (luciferase) and delayed loss of membrane integrity (DISC, EVA/PCD) among others. These improvements led to more biologically relevant measures. However, a second hurdle arose as investigators chose to amplify subculture and propagate tumors into what are now referred to as human tumor organoids. While these 3-D cultures are more reflective of tumor cell-cell interactions found in cancers, they cannot recreate the cell-vasculature, cell-stroma, inflammatory environment nor cancer associated fibroblast interactions with human cancer cells required for accurate response prediction. To address these shortcomings we developed the ex vivo analysis of programmed cell death (EVA/PCD) platform that applies native state tumor explants directly from surgical specimens to select drugs and combinations. To date we have reported 11,680 individual analyses. Positive prospective results have been reported in breast cancer (TTP p &amp;lt; 0.03); ovarian cancer (TTP p = 0.05), lung cancer (2-fold improvement in ORR; TTP p =0.035) and recently ovarian and uterine cancer (DFS p = 0.038) with two meta-analyses confirming the predictive validity in hematologic and solid tumors. With over 20 years of CLIA-approved laboratory experience in the study of human tumor primary culture explants, the EVA/PCD platform provides improved response and time to progression and offers the opportunity to streamline drug development, curtail costs and avoid futile care in patients with advanced and refractory malignancies. Citation Format: Adam J. Nagourney, Steven S. Evans, Paulo D'Amora, Nise Yamaguchi, Paula Bernard, Federico Francisco, Joshua B. Gipoor, Robert Alan Nagourney. Human tumor primary culture analyses for the prediction of response to chemotherapy, targeted agents, drug combinations and metabolic inhibitors: The role of ex vivo analysis of programmed cell death in cancer therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4910.

Multiplication of lavender (L. angustifolia) and lavandin (Lavandula x intermedia) in explant culture

Abstract Lavender plants are preferably propagated vegetatively. The aim of the work was to propose the composition of nutrient media for in vitro multiplication of plants of Lavandula angustifolia (var. Krajová and Beta) and Lavandula x intermedia (var. Budrovka and Grosso). Explant cultures were initiated from apical or nodal segments. Apical segments were more suitable and regenerated the highest number of shoots in MS medium supplemented with 1 mg/L BAP with or without 0.1 mg/L IAA in all used varieties. Nodal segments are suitable for var. Budrovka, where the number of shoots was not statistically different between the apical or nodal segments and among the media tested. Shoot multiplication of L. angustifolia varieties was the most effective in the MS media with 0.5 or 1 mg/L BAP. For variety Grosso, the optimal medium was MS with 1 mg/L BAP, while var. Budrovka regenerated a similar number of shoots in all media supplemented with 0.5 or 1 mg/L BAP with or without 0.1 mg/L IAA. All genotypes rooted with the highest frequency on growth regulator-free medium with a half dose of MS salts. After transplanting into the soil and ex vitro acclimatization, plantlets survivability after 10 weeks was 78.9 – 87.7%, except for var. Beta with a lower survivability of 50%. The proposed procedures enable a rapid plant multiplication of varieties Budrovka, Grosso and Krajová. With the Beta variety, it is advisable to continue working on the optimization of nutrient media to verify whether the procedures can be further optimized.

Ex vivo intervertebral disc cultures: degeneration-induction methods and their implications for clinical translation

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no "one-fits-all" IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside.