Tissue Culture Models of Acute Kidney Injury: From Tubule Cells to Human Kidney Organoids

Acute kidney injury (AKI) affects approximately 13.3 million people around the world each year, causing chronic kidney disease and/or mortality. The mammalian kidney cannot generate new nephrons after postnatal renal damage and regenerative therapies for AKI are not available. Human kidney tissue culture systems can complement animal models of AKI and/or address some of their limitations. Donor-derived somatic cells, such as renal tubule epithelial cells or cell lines (RPTEC/hTERT, ciPTEC, HK-2, Nki-2, and CIHP-1), have been used for decades to permit drug toxicity screening and studies into potential AKI mechanisms. However, tubule cell lines do not fully recapitulate tubular epithelial cell properties in situ when grown under classic tissue culture conditions. Improving tissue culture models of AKI would increase our understanding of the mechanisms, leading to new therapeutics. Human pluripotent stem cells (hPSCs) can be differentiated into kidney organoids and various renal cell types. Injury to human kidney organoids results in renal cell type crosstalk and upregulation of kidney injury biomarkers that are difficult to induce in primary tubule cell cultures. However, current protocols produce kidney organoids that aren't mature and contain off-target cell types. Promising bioengineering techniques, such as bioprinting and "kidney-on-a17 chip" methods, as applied to kidney nephrotoxicity modeling advantages and limitations are discussed. This review explores the mechanisms and detection of AKI in tissue culture, with an emphasis on bioengineered approaches such as human kidney organoid models.

Nanotechnology in Plant Metabolite Improvement and in Animal Welfare

Plant tissue culture plays an important role in plant biotechnology due to its potential for massive production of improved crop varieties and high yield of important secondary metabolites. Several efforts have been made to ameliorate the effectiveness and production of plant tissue culture, using biotic and abiotic factors. Nowadays, the addition of nanoparticles as elicitors has, for instance, gained worldwide interest because of its success in microbial decontamination and enhancement of secondary metabolites. Nanoparticles are entities in the nanometric dimension range: they possess unique physicochemical properties. Among all nanoparticles, silver-nanoparticles (AgNPs) are well-known for their antimicrobial and hormetic effects, which in appropriate doses, led to the improvement of plant biomass as well as secondary metabolite accumulation. This review is focused on the evaluation of the integration of nanotechnology with plant tissue culture. The highlight is especially conveyed on secondary metabolite enhancement, effects on plant growth and biomass accumulation as well as their possible mechanism of action. In addition, some perspectives of the use of nanomaterials as potential therapeutic agents are also discussed. Thus, the information provided will be a good tool for future research in plant improvement and the large-scale production of important secondary metabolites. Elicitation of silver-nanoparticles, as well as nanomaterials, function as therapeutic agents for animal well-being is expected to play a major role in the process. However, nanosized supramolecular aggregates have received an increased resonance also in other fields of application such as animal welfare. Therefore, the concluding section of this contribution is dedicated to the description and possible potential and usage of different nanoparticles that have been the object of work and expertise also in our laboratories.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air–liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

PENGARUH KOMBINASI IAA (Indole-3-Acetic Acid) DAN BAP (6-Benzylaminopurine) TERHADAP INISIASI TANAMAN VANILI (Vanilla planifolia Andrews)

Provision of Vanilla (Vanilla planifolia Andrews) with traditional cultivation often has problems, including the availability of unhealthy seeds due to disease caused by Fusarium oxysporum. This disease can thwart vanilla plantations up to 85% because the pathogen can infect all parts of the vanilla plant making it difficult to control efforts. Alternative efforts were made to overcome this problem, namely through the initiation process in tissue culture by combining the hormones IAA (indole-3-acetic acid) and BAP (6-benzylaminopurine). This study aims to determine the effect and the maximum concentration of the combination of IAA and BAP on the initiation of vanilla plants. This study was conducted based on a completely randomized design (CRD) with 5 treatments and 3 repetitions. This treatment consisted of: A (MS + 0.1 ppm IAA + 1 ppm BAP), B (MS + 0.2 ppm IAA + 1 ppm BAP), C (MS + 0.3 ppm IAA + 1 ppm BAP), D (MS + 0.4 ppm IAA + 1 ppm BAP), and E (MS + 0.5 ppm IAA + 1 ppm BAP). Observations were made after 40 days from the planting process. The results showed that the concentration of the combination of IAA and BAP can affect the growth and organogenesis of the initiation of vanilla plants (Vanilla planifolia Andrews). Treatment C (MS + 0.3 ppm IAA + 1 ppm BAP) was the best concentration from this study, with average values: number of shoots (1), shoot length (1.73 cm), number of roots (1), and number of leaves (1.33).

The Effect of Polyamines and Silver Thiosulphate on Micropropagation of Date Palm Followed by Genetic Stability Assessment

There are some limitations in the practical applications of in vitro date palm tissue culture, such as low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. This study’s objective was to investigate the effect of the two types of polyamines (putrescine "PUT" and spermidine" SPD") in combination with silver thiosulfate (STS) on the growth and development and genetic stability of cultures of Quntar cultivar. Media supplemented with 75 mg L−1 SPD in combination with 10 mgL−1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective in root formation and the number of roots per shoot, where the best result 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mgL−1 PUT and 10 mgL−1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 90% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar is beneficial in improving shoot organogenesis, rooting, and production of genetically stable date palm plants.

Aklimatisasi Anggrek Species Hasil Kultur Jaringan Melalui Pemberdayaan Masyarakat Dusun Gempol

Gempol Village is a village on Mount Ungaran that has made efforts to preserve orchid species. Through mentoring and training from the UNNES Research Team which began in 2011, as well as greenhouse facilitation from PT Indonesia Power, the Gempol village community who are members of the Omah Sawah Community began to make efforts to conserve orchid species. The results of the identification of the problems experienced by community groups as foster partners can be grouped into three aspects, namely knowledge and skills of acclimatization of orchids from plant tissue culture, post-acclimatization management/care, and supporting infrastructure for acclimatization of tissue cultured orchids, where these three aspects are interrelated. The methods used include lecture and question and answer activities, practice, and mentoring. The result achieved is that the orchid species acclimatization activity in Gempol Hamlet, Ngesrepbalong Village has been carried out with satisfactory results. Activities are carried out through training and assistance to community groups who are members of Omah Sawah. The results of the evaluation of the participants showed that the participants' understanding and skills improved after this activity was carried out, even providing ideas for participants to apply to cultivated orchids. The result is enough to generate economic income for the participants, because some of their cultivated orchids are sold. Abstrak Dusun Gempol adalah satu dusun di Gunung Ungaran yang telah melakukan upaya pelestarian anggrek species hutan . Melalui pendampingan dan pelatihan dari Tim Peneliti UNNES yang dimulai pada tahun 201, serta fasilitasi greenhouse dari PT Indonesia Power, masyarakat Dusun Gempol yang tergabung dalam Komunitas Omah Sawah mulai melakukan upaya pelestarian anggrek species. Hasil identifikasi terhadap permasalahan yang dialami kelompok masyarakat sebagai mitra binaan dapat dikelompokkan menjadi tiga aspek, yaitu pengetahuan dan keterampilan aklimatisasi anggrek hasil kultur jaringan tanaman, pengelolaan/ perawatan pasca aklimatisasi, dan sarana prasarana penunjang aklimatisasi anggrek hasil kutur jaringan, dimana ke tiga aspek ini salingterkait. Metode yang dilakukan, meliputi kegiatan ceramah dan tanya jawab, praktik, dan pendampingan. Hasil yang dicapai adalah kegiatan aklimatisasi anggrek species di Dusun Gempol Desa Ngesrepbalong telah dilaksanakan dengan hasil yang memuaskan. Kegiatan dilaksanakan melalui pelatihan dan pendampingan terhadap kelompok masyarakat yang tergabung di Omah Sawah. Hasil evaluasi terhadap peserta menunjukkan bahwa pemahaman dan ketrampilan peserta meningkat setelah dilakukan kegiatan ini, bahkan memberikan ide bagi peserta untuk menerapkan pada anggrek budidaya. Hasilnya cukup membuahkan pemasukan ekonomi bagi peserta, karena beberapa anggrek budidaya mereka laku dijual.

Low CO2 Levels Are Detrimental for In Vitro Plantlets through Disturbance of Photosynthetic Functionality and Accumulation of Reactive Oxygen Species

Photosynthesis of plantlets in tissue culture containers is not considered important, compared to photosynthesis of ex vitro plants, due to the exogenous source of carbohydrates present in tissue culture media. However, CO2 starvation can generate a burst of reactive oxygen species (ROS). We examined this phenomenon in tissue culture, since CO2 levels may become very low during the light period. The research was carried out with lily scales, regenerating adventitious bulblets, and with Arabidopsis seedlings. CO2 starvation was achieved by placing a small vial of concentrated KOH solution in the culture container. CO2 removal reduced the growth of regenerated lily bulblets by 33% or 23%, with or without sucrose in the medium, respectively. In Arabidopsis seedlings, CO2 removal decreased growth by 50% or 78% in the presence or absence of sucrose in the medium, respectively. Therefore, the addition of sucrose as a replacement for photosynthesis resulted in only partial recovery of growth. Staining with nitroblue tetrazolium (NBT) showed little to no ROS in ex vitro growing seedlings, while abundant ROS were detected in seedlings grown under in vitro CO2 starvation. Seedlings grown under normal tissue culture conditions (no CO2 withdrawal) showed low levels of ROS. In lily tissue culture, CO2 starvation decreased the maximum quantum efficiency of photosystem II (Fv/Fm) from 0.69 to 0.60, and in Arabidopsis from 0.76 to 0.62. Fv/Fm of ex vitro lily and Arabidopsis seedlings was 0.77 and 0.79, respectively. This is indicative of a disturbance in photosynthesis functionality and the occurrence of in vitro stress under reduced CO2 concentrations. We conclude that poor growth, in the absence of CO2, was partly due to strongly reduced photosynthesis, while the detrimental effects were most likely due to a burst of ROS.

Gene expression programs during callus development in tissue culture of two Eucalyptus species

Background Eucalyptus is a highly diverse genus of the Myrtaceae family and widely planted in the world for timber and pulp production. Tissue culture induced callus has become a common tool for Eucalyptus breeding, however, our knowledge about the genes related to the callus maturation and shoot regeneration is still poor. Results We set up an experiment to monitor the callus induction and callus development of two Eucalyptus species - E. camaldulensis (high embryogenic potential) and E. grandis x urophylla (low embryogenic potential). Then, we performed transcriptome sequencing for primary callus, mature callus, shoot regeneration stage callus and senescence callus. We identified 707 upregulated and 694 downregulated genes during the maturation process of the two Eucalyptus species and most of them were involved in the signaling pathways like plant hormone and MAPK. Next, we identified 135 and 142 genes that might play important roles during the callus development of E. camaldulensis and E. grandis x urophylla, respectively. Further, we found 15 DEGs shared by these two Eucalyptus species during the callus development, including Eucgr.D00640 (stem-specific protein TSJT1), Eucgr.B00171 (BTB/POZ and TAZ domain-containing protein 1), Eucgr.C00948 (zinc finger CCCH domain-containing protein 20), Eucgr.K01667 (stomatal closure-related actinbinding protein 3), Eucgr.C00663 (glutaredoxin-C10) and Eucgr.C00419 (UPF0481 protein At3g47200). Interestingly, the expression patterns of these genes displayed “N” shape in the samples. Further, we found 51 genes that were dysregulated during the callus development of E. camaldulensis but without changes in E. grandis x urophylla, such as Eucgr.B02127 (GRF1-interacting factor 1), Eucgr.C00947 (transcription factor MYB36), Eucgr.B02752 (laccase-7), Eucgr.B03985 (transcription factor MYB108), Eucgr.D00536 (GDSL esterase/lipase At5g45920) and Eucgr.B02347 (scarecrow-like protein 34). These 51 genes might be associated with the high propagation ability of Eucalyptus and 22 might be induced after the dedifferentiation. Last, we performed WGCNA to identify the co-expressed genes during the callus development of Eucalyptus and qRT-PCR experiment to validate the gene expression patterns. Conclusions This is the first time to globally study the gene profiles during the callus development of Eucalyptus. The results will improve our understanding of gene regulation and molecular mechanisms in the callus maturation and shoot regeneration.