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Rakovina Therapeutics Provides Update on Previously Announced Private Placement
BOSTON, July 22, 2024 (GLOBE NEWSWIRE) -- TransCode Therapeutics, Inc. (Nasdaq: RNAZ), (“TransCode” or the “Company”), a clinical-stage RNA oncology company committed to more effectively treating cancer using RNA therapeutics, today announced the pricing of a public offering of 10,000,000 shares of its common stock at a public offering price of $0.30 per share, for expected gross proceeds of $3,000,000, before deducting placement agent commissions and offering expenses. All of the shares of common stock are being offered by the Company. The offering is expected to close on July 24, 2024, subject to satisfaction of customary closing conditions.
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A seventh person has essentially been cured of HIV after receiving a stem cell transplant nearly a decade ago, doctors announced Thursday.
The 60-year-old unidentified German man was suffering from acute myeloid leukemia when he underwent the risky procedure to replace his unhealthy bone marrow in October 2015.
He quit taking anti-retroviral drugs which stop HIV from reproducing in September 2018. He remains in viral remission and appears to be cancer-free.
A healthy person has many wishes, a sick person only one, the man, who wishes to remain anonymous, said of his progress.
Dr. Christian Gaebler, a physician-scientist at the Charit-Universittsmedizin Berlin, is slated to present the case next week at the 25thInternational AIDS Conference.
The longer we see these HIV remissions without any HIV therapy, the more confidence we can get that were probably seeing a case where we really have eradicated all competent HIV, Gaebler said.
At a news conference last week, International AIDS Society President Sharon Lewin cautioned against using the word cure.
Still, she said, being in remission for more than five years means he would be close to being considered cured.
There is one major difference between the German mans case and most of the rest.
Five of the other six patients received stem cells from donors with two copies of a rare genetic mutation that stops HIV from replicating.
The German patient is said to be the first to have received stem cells from a donor with just one copy of the mutated gene and he had a copy of the gene himself.
About1% of Caucasians have two copies of thedefective gene, while 10% to 18% of people with European heritage are estimated to have one copy of the gene, thus expanding the potential donor pool.
Some 39 million people around the world are living with HIV, the virus that causes AIDS. Very few will be able to access this treatment, as it is reserved for those with HIV and aggressive leukemia.
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7th person likely cured of HIV in a remarkable case - New York Post
Jul 19, 2024 14:00:00
A German HIV patient has been symptom-free for six years after undergoing a stem cell transplant, making him likely the seventh person to be essentially cured of HIV after receiving a stem cell transplant, doctors have announced.
Seventh person likely 'cured' of HIV, doctors announce
According to the research abstract, the German man in long-term remission was first diagnosed with HIV in 2009, underwent a bone marrow transplant for leukemia in 2015, and then stopped taking antiretroviral drugs to reduce the amount of HIV in his blood in 2018. Nearly six years have passed since then, and he has been deemed to have achieved long-term remission as he has not developed HIV or cancer.
'We can never be absolutely certain that the last traces of HIV have been eradicated,' said Dr. Christian Gabler of Charit University Hospital, who treated the patient. 'However, this patient's case is a strong indicator of HIV cure.'
'Researchers are hesitant to use the word 'cure' because it's not clear how long cases like this need to be followed for, but more than five years in remission means this man is 'near cure,'' said Sharon Lewin, president of the International AIDS Society.
Lewin said there's an important difference between this man's case and other HIV patients who have achieved long-term remission: All but one of them reportedly received stem cells from donors who carried a rare mutation that stops HIV from entering the body's cells.
All of these donors were found to have a defective CCR5 gene, meaning they had inherited two copies of the mutated gene (one from each parent), which gave them 'intrinsic immunity to HIV,' Lewin said.
However, this patient is the first to have received stem cells from a donor who inherited only one mutated gene.
While only 1% of Europeans have inherited two copies of the mutated CCR5 gene, as many as 15% have only one, which suggests that more donors may be accepted in the future, the researchers said.
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Doctors say 60-year-old German man likely to become seventh person in the world cured of HIV - GIGAZINE
Animal ethics
All of the animal studies were approved by the Animal Care Committee at the University of Manitoba (protocol AUP-22-005).
The HDMB03 and MB3W1 group 3 MB cell lines were kindly provided by Milde et al.53 and Wlfl and colleagues54, respectively. Short tandem repeat profiling (American Type Culture Collection) was used to authenticate cell lines in 2021. For tumoursphere formation, both cell lines were dissociated into single-cell suspensions. HDMB03 cells were then resuspended in StemPro NSC Serum-Free Medium (Life Technologies) and MB3W1 cells were resuspended in stem cell media (DMEM/F-12, 2% B27, 1% MEM Vitamin Solution, 20ngml1 basic fibroblast growth factor, 20ngml1 epithelial growth factor, 40Uml1 penicillin and 40gml1 streptomycin). Suspensions were plated at clonal density (for HDMB03, three cells per l; for MB3W1, five cells per l) in either 6- or 24-well ultra-low-attachment plates. Tumourspheres were incubated undisturbed at 37C under 5% CO2 for either 3 or 5d, counted and then measured. Tumoursphere number (5d), tumoursphere size (3 or 5d), cell viability (3d) and total cell number (5d) were evaluated. ImageJ (Fiji) was used to evaluate individual tumoursphere size. The results were displayed as the cumulative frequency distribution of the tumoursphere area. Only tumourspheres greater than 25m in diameter were included in these analyses55. For morpholino treatment, a custom splice-blocking morpholino to induce exon skipping was designed for PPHLN1 (ENSG00000134283.13, exon 6; 5-AGAGTCTAGCTCAAAACTCACCTCT-3) and MADD (ENSG00000110514.14, exon 26; 5-GTCCCTTCCTGCCAATTTGAGAGCA-3). Concentrations of 1, 5 or 10M in double-distilled water were added to single-cell suspensions and then cultured in ultra-low-attachment plates. An Annexin V Apoptosis Detection Kit (Annexin V-PE; BD Biosciences) was used to evaluate cell death, as previously described15,56.
Silencer Select siRNAs 9931 and 9932 (Life Technologies) were used at 30nM to knockdown OTX2 expression in group 3 cells, as previously described13,15. A scramble (non-silencing) sequence was used as a negative control. For bulk RNA-seq, OTX2 was silenced in tumourspheres from three independent biological replicates of HDMB03 and MB3W1 cells and silencing was confirmed by western blot 3d following transfection, as described13,15. Total RNA was previously extracted using a Norgen RNA extraction kit (Norgen Biotek) followed by bulk RNA-seq (paired end; 150base pairs) targeting 100million total reads for adequate depth of coverage for splicing analyses. Sequencing was performed at the Ottawa Hospital Research Institute/StemCore laboratories as described3. The raw data generated were previously deposited in the Gene Expression Omnibus (GEO) database under the access code GSE189238.
Raw reads were mapped to the human genome using the hg19 reference with the STAR aligner (version 2.5.2b)57. Picard tools and SAMtools were used to remove PCR-duplicated reads and low-mapping-quality reads (mapping quality<20), respectively. rMATS (version 3.0.9)58an event-based toolwas used to identify differentially spliced events. We analysed five distinct alternative splicing events using rMATS: mutually exclusive exons, retained introns, skipped exons, alternative 3 splice sites and alternative 5 splice sites. Briefly, rMATS features a count-based model that calculates PSI scores among replicates using spliced reads and reads that map to the exon body. To call statistically significant splicing events, each splicing event had to: (1) have a PSI of >10%; (2) be supported by at least 15 unique reads; and (3) have an FDR of <0.05. For gene expression analysis, aligned reads were counted using htseq-count version 0.6.1p1 (ref. 59) and normalized according to the DESeq size factors method60. To call DEGs, we used a fold change of 2 and an FDR of <0.05. Statistical analysis was performed and plots were generated using R environment version 3.6.0. Sashimi plots were generated using ggsashimi.py script61 with the parameter --min-coverage 5. We performed motif analyses to evaluate the enrichment of RBP motifs enriched near OTX2-regulated ASEs using a compilation of 110 known RBP motifs from the literature27,28. Briefly, we used rMAPS62 to identify known RBP motifs that were significantly enriched in OTX2-regulated ASEs compared with control exons (background). rMAPS examines adjacent intronic and exonic sequences and counts the number of times the motif matches each sequence to compute an enrichment score.
To explore the splicing landscape of group 3 and group 4 MB, we examined previously published bulk RNA-seq results for group 3 and group 4 tumours3. Briefly, we excluded exons that were not expressed in at least 25% of samples and excluded 5% of the total number of samples that had the least number of detected exons. We performed correlation-based analysis between OTX2 expression and PSI values and applied FDR correction. We considered statistically significant exons that had an adjusted Pvalue of <0.01.
To determine whether OTX2-regulated exons recapitulate the developmental origins of group 3 and group 4 tumours, we used previously published cerebellar bulk RNA-seq data6,31 for the rhombic lip (RLVZ and RLSVZ at post-conception weeks (PCWs) 9 and 10; n=4) and EGL (at PCW 15; n=4). We also compared the splicing profile of each group 3 and 4 MB tumour with that of the human rhombic lip (pooled RLVZ and RLSVZ at PCWs 9 and 10; n=4). We applied the same filters as described above. Because the depth of coverage for each tumour can impact the number of significant ASEs, only exons that were expressed in all tumours were retained (an event needs to be supported by at least 15 unique reads) for subsequent analyses. Briefly, the list of exons expressed in each tumour was extracted. Then, we removed samples that had too few or too many expressed exons and considered them as outliers (samples that were outside box plot whiskers: n
For RNA-seq on PPHN1-Mo-treated cells, HDMB03 cells were treated with 1M Ctrl-Mo or PPHLN1-Mo for 5d. Cells were collected and RNA was extracted using a Total RNA purification kit (Norgen Biotek). RNA-seq was performed as described above using the same alignment parameters and analysis tools.
We performed functional and pathway enrichment analysis on genes exhibiting significant changes in expression following OTX2 silencing; this was done separately for genes up- and downregulated following knockdown. Enrichment analysis was performed using the ClusterProfiler tool65 to look at the enrichment of Gene Ontology molecular function and biological process annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Within each group of annotations, enrichment was calculated by comparison with the hypergeometric distribution (equivalent to a one-tailed Fishers exact test). Multiple testing correction was performed using the BenjaminiHochberg approach. The KEGG pathway data were obtained from the KEGG website on 11 February 2022.
The DSGs PPHLN1, PAPOLA, MADD and MBD1 were selected for further validation by RT-PCR. Total RNA was extracted from both HDMB03 and MB3W1 tumourspheres using a Total RNA Purification Kit (Norgen Biotek). First-strand complementary DNA was generated using the SuperScript III First-Strand Synthesis kit (Life Technologies). PCR was performed using the MiniAmp Plus Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific) with the following parameters: 98C for 10s, 55C for 5s and 72C for 5s over 3035 cycles. The primer sequences for each gene evaluated are listed in Supplementary Table 16. GAPDH was used as a loading control for morpholino validation. Samples were run on 2% agarose gels and imaged on a Fusion FX Vilber Lourmat chemiluminescence imaging system.
Gene silencing of LASR complex members and select DSGs was performed using Silencer Select siRNAs (Life Technologies) at 30nM (HDMB03) or 15nM (MB3W1) concentrations. A non-silencing scramble siRNA was utilized as a negative control. siRNA sequences for all genes are provided in Supplementary Table 17. Gene silencing was validated by immunoblotting. A list of the antibodies used for validation studies is provided in Supplementary Table 18.
HDMB03 cells were seeded at 2105cells per well under stem cell conditions and cultured for 72h. Cells were then fixed and quenched using 1% paraformaldehyde and 0.125M glycine, respectively. Lysates were next sonicated to 100500base pair fragments followed by incubation overnight with primary antibodies (Supplementary Table 18) at 4C. Protein A Dynabeads (Invitrogen) were used to capture antibody/chromatin complexes for 4h followed by washing, elution and reverse crosslinking. Chromatin was purified using the Qiagen PCR Purification Kit. Chromatin was amplified using primers designed to flank known OTX2 binding sequences (Supplementary Table 16) and GoTaq qPCR master mix (Promega). Fold enrichment of OTX2 binding was calculated and graphed with Prism.
Wild-type and homeodomain-deleted human OTX2 sequences cloned into the plasmid pcDNA3.1+ were purchased from GenScript. The coding sequences were amplified by PCR, adding overlap sequences to the 5 ends of primers to allow NEBuilder HiFi assembly (New England Biolabs) with NheI/ClaI-linearized pCAG-H2B-mAG-P2A-3XHA-TurboID3, yielding pCAG-H2B-mAG-P2A-3XHA-hOTX2 and pCAG-H2B-mAG-P2A-3XHA-hOTX2HD. All cloning reagents were purchased from New England Biolabs. The sequences of the resultant plasmids were verified by whole-plasmid sequencing (Eurofins Genomics).
Single-nucleus RNA-seq data from the developing human cerebellum were obtained and processed as previously described31. Briefly, sample fastqs were aligned to the human reference genome hg19 and the resultant count data were interpreted in Seurat (version 4.1.0)66 using the R environment (version 4.1.3) in order to perform quality control, normalization by SCTransform67, dimensionality reduction (uniform manifold approximation and projection), clustering (shared nearest neighbour and Louvain modularity optimization) and cell type identification. Trajectory inference within the developing human glutamatergic cells was also performed as previously described3. Briefly, Slingshot (version 1.6.1)68 was used to find the expected granule neuron and UBC lineages and TradeSeq (version 1.2.01)69 was used to statistically correlate gene expression with lineage pseudotime values. The resulting significant lineage-associated genes were then used in downstream analyses.
Proteinprotein interactions (PPIs) within both the OTX2-regulated DSGs list and the OTX2 TurboID hits list were determined by querying the STRING database (version 11.5)70. Briefly, gene lists were inputted and the resulting networks of PPIs were further filtered for only known physical interactions (Experiments=T; Databases=T) with confidence scores of at least 0.150 (low confidence) or 0.400 (medium confidence), where indicated. This list of known PPIs was visualized using Cytoscape (version 3.8.0)71. Nodes were coloured by relevant enriched pathways in the queried lists using g:Profiler72 with the Gene Ontology Biological Processes (GO:BP), KEGG, Reactome and WikiPathways databases and an FDR threshold of 0.1. A full list of enriched pathways is provided in Supplementary Table 2. Nodes were manually rearranged to prevent node overlap; thus, the edge length is not representative of any value.
Protein quantities (1025g) were loaded onto 1012% Tris-glycine gels and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transferral to nitrocellulose membranes, blocking with 5% milk and incubation overnight with select primary antibodies (details provided in Supplementary Table 18). Secondary antibodies conjugated to horseradish peroxidase were added for 1h at room temperature followed by detection using SuperSignal West Pico. A Fusion FX Vilber Lourmat chemiluminescence imaging system was used to capture the images.
HDMB03 cells were plated at 5104cells per well in 24-well plates on glass coverslips that had been coated in poly-d-lysine (Thermo Fisher Scientific) for 10min before being washed with molecular-grade H2O. For OTX2 silencing, HDMB03 cells were treated with scrambled RNA (negative control) or Silencer Select siRNA 9931 (Supplementary Table 17). HDMB03 cells were transfected with OTX2WT or OTX2HD plasmid (the latter resulting in deletion of the DNA-binding homeodomain) 24h after plating, as described. At 72h after HDMB03 cells were plated, they were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 30min. Cells were then washed three times with PBS before permeabilization with 0.1% Triton X-100 in PBS for 20min at room temperature. After washing three times with PBS, cells were blocked with 3% lamb serum/PBS for 1h at room temperature, followed by replacement of the blocking buffer with 1:100 primary antibody (RBFOX2, HNRNPC or HNRNPM) (Supplementary Table 18) in 1% lamb serum/PBS. After 1h incubation at room temperature, the cells were washed three times with PBS before incubating with either anti-rabbit (RBFOX2 and HNRNPM) or anti-mouse (HNRNPC) secondary antibody. For the OTX2 silencing and OTX2 deletion mutation experiments, Alexa Fluor 488 and Alexa Fluor 594 were used, respectively (Supplementary Table 18), at a dilution of 1:1,000 for 1h at room temperature. Cells were then washed three times with PBS and once with H2O, followed by mounting with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories). This was performed by placing a drop of mounting medium on a glass slide and flipping the coverslip with the cells onto the drop of mounting medium. Images were taken at 40 magnification using an EVOS M5000 Imaging System and the cytoplasmic area per cell was quantified using CellProfiler73.
TurboID experiments were performed as previously described by Cho et al.74. NEBuilder HiFi DNA Assembly (New England Biolabs) was used to introduce the human OTX2 sequence into the plasmid pCAG-H2B-mAG-P2A-3xHA-TurboID3. The vector was linearized with KpnI and ClaI and assembled with PCR-amplified 3xHA-TurboID (FW_KpnI_Turbo and RV_Turbo_GGSGG primers) and PCR-amplified hOTX2 (using reverse-transcribed RNA (Norgen) recovered from HDMB03 cells and RNEB_ClaI_N-Otx2 and FNEB_Link_N-OTX2 primers) to generate pCAG-H2B-mAG-P2A-3xHA-TurboID-OTX2. PCR-amplified sequences in the final plasmid were verified by Sanger sequencing. The primers used were as follows: FW_KpnI_Turbo (5-AGAACCCTGGACCTGGTACCATGTACCCGTATGATGTTCCGG-3); RV_Turbo_GGSGG (5-GCCTCCAGATCCGCCCTTTTCGGCAGACCGCAGACTG-3); RNEB_ClaI_N-OTX2 (5-CGAGCTCTAGATCATCGATTTACAAAACCTGGAATTTCCACGAGGATGTCTG-3); and FNEB_Link_N-OTX2 (5-GGCGGATCTGGAGGCATGATGTCTTATCTTAAGCAACCGCCTTAC-3).
For the TurboID transfection studies, 5105 HDMB03 cells per well were seeded into 6-well ultra-low attachment plates and transfected using Lipofectamine 3000 (Thermo Fisher Scientific) with 12g plasmid, as previously described by Hendrikse et al.3. Tumourspheres were cultured for 72h in StemPro Medium (Life Technologies) and then treated with 500M biotin-d (B4639; SigmaAldrich) for 15min. Tumourspheres were then washed in 1 PBS and lysed with 1ml 1 RIPA (20mM Tris-HCl (pH7.5), 150mM NaCl, 1mM EDTA-Na2, 1mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5mM sodium pyrophosphate, 1mM -glycerophosphate, 1mM Na3VO4 and 1 Halt Protease and Phosphatase Inhibitor (Thermo Fisher Scientific)) for 10min3. Samples were then sonicated over 210s pulses at 30% load to shear DNA (an FB50 sonicator with microprobe; Thermo Fisher Scientific) and clarified by centrifugation at 16,000g and 4C for 10min. Supernatants were quantified using the Bradford protein assay. Streptavidinsepharose bead slurries (50l aliquots) were washed once in 1ml 1 RIPA. Washed beads were resuspended in 1ml 1 RIPA containing 2mg protein supernatant and rotated at 4C overnight. The next day, samples were centrifuged for 5min at 1,000g and bead pellets were washed twice in 1ml 2% sodium dodecyl sulfate solution (in double-distilled water) and then three times (8min each time) in 1ml Wash Buffer (50mM Tris-HCl (pH7.4) and 8M urea) at room temperature. Samples were resuspended in Storage Buffer (285l of ammonium bicarbonate (50mM) and 15l of 1mM biotin) and stored on ice.
An Orbitrap Exploris 480 instrument (Thermo Fisher Scientific) was used to obtain mass spectrometry data, as described by Hendrikse et al.3 Briefly, all mass spectrometry raw files were processed with Proteome Discoverer (version 2.20.388) at the Manitoba Centre for Proteomics and Systems Biology. The files were searched for tryptic peptides against the human UniProt protein database (December 2020) using SEQUEST with standard Orbitrap settings, as previously described3. In addition, up to two missed cleavages were permitted, with a parent and fragment mass tolerance of 0.02Da and 15ppm. A fixed modification of cysteine carbamidomethylation was applied. Also, variable modifications including deamidation (at Asn and Gln), amino-terminal acetylation, oxidation (at Met and Trp), phosphorylation (at Ser, Thr and Tyr), ubiquitylation (at Lys), double oxidation (at Met and Trp) and biotinylation (at Lys) were permitted. The results were filtered by 1% FDRs at both the peptide and protein levels. SAINTexpress (version 1.0.0)47,48 was used to calculate the probability of each potential proximal protein interaction from background contaminants using default parameters (n=4 OTX2 biological replicates and n=3 control biological replicates). A complete list of OTX2-interacting proteins is provided in Supplementary Table 1.
HDMB03 MB cells were grown as tumourspheres for 3d, dissociated and lysed with immunoprecipitation lysis buffer (25mM Tris (pH7.4), 150mM NaCl, 1% Triton X-100 and 5mM EDTA) plus 1 protease inhibitor complex. Samples were then pre-cleared for 2h and rotated at 4C. Immunoprecipitation was carried out overnight with 2g OTX2 antibody or immunoglobulin G. For co-IP on HDMB03 cells overexpressing wild-type OTX2 or OTX2 lacking its DNA-binding homeodomain, immunoprecipitation was carried out with a haemagglutinin antibody. Magnetic beads were blocked with 1% bovine serum albumin solution for 1h, washed and then added to the protein/antibody complexes for 3h with rotation at 4C. Beads were then washed five times with immunoprecipitation lysis buffer. Complexes were eluted by boiling for 5min in 1 sodium dodecyl sulfate sample buffer. Interaction with LASR complex members (RBFOX2, ILF2, ILF3, MATR3, DDX5, HNRNPM, HNRNPC and HNRNPH) was then determined by immunoblotting. The antibody conditions are described in Supplementary Table 18.
Subcellular fractionation was performed using previously established protocols17,30. HDMB03 cells were lysed in cytoplasmic lysis buffer (CLB; 340mM sucrose, 10mM Tris-HCl (pH7.9), 0.1mM EDTA, 3mM CaCl2, 1mM dithiothreitol (DTT), 2mM MgCl2, 0.5% NP-40 and protease and phosphatase inhibitors) on ice for 10min and the cytoplasmic fraction was removed following centrifugation at 3,500g for 15min. Cellular pellets were then washed with CLB wash buffer (CLB lacking NP-40) followed by centrifugation at 800g for 3min. Soluble nuclear fractions were removed by lysing the cells for 5min on ice with nuclear lysis buffer (10% glycerol, 3mM EDTA, 20mM HEPES (pH7.9), 150mM KOAc, 1.5mM MgCl2, 1mM DTT, 0.1% NP-40 and protease and phosphatase inhibitors) followed by centrifugation at 15,000g for 30min. The RNA fraction was then isolated by lysing cells with nuclease incubation buffer (10% glycerol, 150mM HEPES (pH7.9), 1.5mM MgCl2, 1mM DTT, 150mM KOAc and protease and phosphatase inhibitors) plus 10mgml1 RNase, whereas the DNA fraction was isolated by lysing cells with nuclease incubation buffer plus DNAse I (1U) for 30min at 37C. RNA and DNA fractions were then recovered by centrifugation at 20,000g for 30min. Samples were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the antibodies described in Supplementary Table 18.
For the in vivo morpholino studies, HDMB03 and MB3W1 cells were treated with 1 or 2.5M control or PPHLN1-Mo for 5d in culture. Cells were dissociated and 1105 (HDMB03) or 2105 (MB3W1) cells were injected into the cerebellums of 7- to 9-week-old male NOD-SCID mice. NOD-SCID mice were housed in individually ventilated cages (Tecniplast). Irradiated feed was used and bedding was sterilized by steam autoclave. The room ambient temperature was 2123C with a relative humidity target of 50% (the range was 3060%). Beginning at 06:00am, the light cycle was 12h on and 12h off. The endpoint was defined as a 20% weight reduction from peak body weight and/or significant ataxia and ruffled fur. MRI was performed on a cryogen-free FlexiScan 7T system (MR Solutions). For tumour size calculations from MRI images, the ImageJ freehand tool was used to determine the volume from 18 serial sections (300m thickness) from each sample. The sum of all of the slices was then used to calculate an overall tumour volume, as previously described55. At the endpoint, tumours were prepared for immunohistochemistry using a mitochondria-specific antibody to detect human cells as well as SOX2 using methods previously described15. The primary and secondary antibody sources and concentrations are listed in Supplementary Table 18.
One Ctrl-Mo and one PPHLN1-Mo formalin-fixed, paraffin-embedded xenograft tumour sample were processed following the GeoMx DSP Slide Preparation User Manual (MAN-10087-04). Briefly, slides were baked at 60C for at least 1h, deparaffinized using CitriSolv d-limonene and then rehydrated. Antigen retrieval was performed using 1 citrate buffer at a pH of 6.0 in a pressure cooker for 15min at 100C on high pressure. Slides were blocked using NanoString blocking buffer W for 1h. Slides were then incubated overnight at 4C with the following ultraviolet-photocleavable, barcode-conjugated antibody panels against a total of 43 targets and six control targets from NanoString Technologies: GeoMx Neural Cell Profiling Panel Human Protein Core for nCounter; GeoMx Cell Death Panel Human Protein Module for nCounter; GeoMx PI3K/AKT Signaling Panel Human Protein Module for nCounter; and GeoMx MAPK Signaling Panel Human Protein Module for nCounter. At the same time, the samples were incubated with morphology antibodies consisting of a 1:100 dilution of Ki67Alexa Fluor 647 (12075S; Cell Signaling Technology) and a 1:500 dilution of MAP2Alexa Fluor 532 (NBP1-92711AF532; Novus Biologicals). The slides were washed and stained with SYTO 13 (S7575; Thermo Fisher Scientific) for 15min before loading onto a GeoMx DSP microscope (NanoString Technologies). Fluorescence images were scanned at 20 and regions of interest (ROIs) along the tumour border and in the tumour core were selected. Oligos from antibodies were cleaved and collected into 96-well plates. Oligos were dried down completely by incubating overnight at room temperature with a permeable plate seal and then rehydrated and hybridized with NanoString HybCode barcodes. Samples were hybridized overnight for at least 16h in a thermal cycler at 67C with a heated lid at 72C. The resulting oligos hybridized to barcode tags were detected and counted using an nCounter Prep Station and Digital Analyzer (NanoString Technologies) the following day. The digital counts of each antibody for each ROI were generated for data analyses and analysed using NanoString GeoMx software. The software has built-in quality control analysis and three ROIs that did not pass quality control metrics due to high binding density were excluded from downstream analysis. The data were normalized by scaling to the negative control immunoglobulin G probes, constituting a signal-to-noise ratio. Counts were further scaled to the number of nuclei per ROI and the normalized counts from the Ctrl-Mo and PPHLN1-Mo samples were calculated.
Prism 8.0 software (GraphPad) was used for all of the statistical analyses. No data were excluded from the analyses. NOD-SCID mice for the animal studies were randomly assigned into treatment groups; however, the remaining experiments were not randomized. Blinding was performed during MRI imaging and tissue preparation for immunohistochemistry. For all of the other experiments, the investigators were not blinded. No statistical methods were used to predetermine sample size, but our sample sizes are similar to those reported in previous publications15,55. BrownForsythe tests were performed to assess the homogeneity of variances for cell culture-based studies. The statistical tests used for all of the experiments are noted in the figure captions.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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Purification technologies for induced pluripotent stem cell therapies - Nature.com
A recent clinical study by Pipeleers and colleagues has brought the possibility of a stem-cell based cure one step closer1. This perspective will summarize the major hurdles that have been overcome to deliver cell-based improvements in glucose control and highlight the key issues that stand between this important proof-of-concept clinical study and a durable cure for the majority of patients living with T1D.
The autoimmune destruction of pancreatic cells creates a lifelong dependence on insulin to control blood sugar levels in individuals with type 1 diabetes (T1D). Over time, poorly managed T1D causes microvascular and macrovascular complications that significantly impact quality of life2. Unfortunately, intensive glucose lowering therapy to reduce these long-term complications of hyperglycemia is accompanied by an increased risk of hypoglycemic events3. Technological solutions aiming to replace cell function with an artificial pancreas can improve glucose control by integrating continuous glucose monitoring with automated insulin delivery4. However, these systems have not yet matched the exquisite blood glucose control provided by human islets5, and T1D patients remain burdened with the ongoing management and expense of a chronic disease.
Therapeutic approaches aimed at restoring a functional -cell mass could eventually eliminate the need for exogenous insulin. Indeed, transplant of cadaveric islets into immunosuppressed T1D recipients has shown that excellent glucose control can be achieved6, while simultaneously reducing hypoglycemic risk7. The benefits of islet transplant to individual T1D islet recipients should not be minimized, however, the limited supply of donor tissue constrains the potential impact of this strategy, which is still only available to clinical trial participants in many countries including the USA. In contrast, human pluripotent stem cells (hPSCs)8,9 could theoretically be expanded and differentiated to restore a functional -cell mass in all eligible patients with T1D if they can be shielded from autoimmune attack.
Initially, a major goal was to optimize stem cell differentiation protocols to produce glucose-responsive cells from hPSCs. The first major success was guided by developmental studies from diverse model organisms10, in which step-wise modulation of key developmental signals produced cells capable of expressing insulin11, albeit at low levels and in a largely constitutive manner. Nevertheless, this was a remarkable demonstration that hPSCs have the potential to be used for cell-replacement therapy. Extensive empirical optimization and an appreciation of the functional importance of islet structure led to -cells with improved function12,13. We note, the in vitro generation and characterization of stem-cell derived islets has been recently reviewed14. However, the observation that in vitro differentiated hPSC-derived -cells exhibit immature physiological responses15, like many other hPSC-derived cell products16, led to consideration of alternative strategies. A surprisingly effective approach has involved halting in vitro differentiation once pancreatic fate is established at the multipotent pancreatic progenitor (PP) stage and allowing -cell differentiation and functional maturation to be guided by endogenous cues post-transplant17. An added benefit of this approach is that PP differentiation is amenable to the large-scale expansion and Good Manufacturing Practice (GMP) production and quality control required for clinical application18. Interestingly, further differentiation and enrichment of hormone-positive islet-like cells prior to transplant does not reduce the in vivo maturation time19.
Now that a suitable cell-source is available, preventing graft rejection is one of the greatest challenges facing hPSC-based therapies. The autoimmune nature of T1D poses a challenge for cell-based therapies since the immune system is poised to destroy newly transplanted material, even if it is derived from the patients own stem cells. As seen with cadaveric transplants, systemic immunosuppression can protect and maintain unmatched donor cells in a functional state6. Furthermore, clinical transplants have shown that ~10,000 islet equivalents/kg provide a functional -cell mass that can eliminate the need for exogenous insulin20, setting a clear goal for therapeutic effect. Unfortunately, this blunt force approach trades dependence on insulin for continuous immunosuppression, which brings increased risks of infections, certain cancers and regimen-specific toxicities21.
Encapsulating transplants in biocompatible materials that prevent immune infiltration, while permitting sufficient diffusion of nutrients and waste products to support -cell health, has been pursued to eliminate the need for systemic immunosuppression. Despite the demonstration over 40 years ago that microencapsulation is sufficient to preserve islet function for several weeks in an animal model without immune suppression22, maintaining a functional -cell mass within cell-impermeable materials remains a major challenge. Microencapsulated islets (single islets or small clusters) can disperse into the recipient tissue where they benefit from a large contact area with the host. However, the impermeable barrier prevents direct contact with blood vessels, which produce a basement membrane that is likely essential for optimal -cell function23. These problems have been even more pronounced in cell-impermeable macroencapsulation devices, where elaborate designs such as intravascular hollow fibers are used to increase exposure to the bloodstream24. However, despite the theoretical advantages of close contact with the blood stream, the serious risk of blood clots associated with vascular prostheses has impeded clinical translation of intravascular devices25. The strengths and weaknesses of additional islet encapsulation technologies have been recently reviewed26.
Because cell impermeable materials necessarily prevent direct contact between -cells and the endothelium, some groups have gone a different direction with cell-permeable devices, including Viacyte with the VC-02 device. Although the exact configuration of the VC-02 remains proprietary, key features that appear to have contributed to clinical success are a perforated encapsulation membrane that is encased in another layer of perforated non-woven fabric27. The VC-02 device loaded with hPSC-derived Pancreatic Endoderm Cells (PECs) that are partially differentiated to the PP stage has been coined the PEC-Direct (Fig. 1).
Partially differentiated hPSC-derived PECs were loaded into devices that mature under the protection of systemic immunosuppression in T1D patients. The perforated design facilitates the infiltration of endothelial cells, while the external non-woven fabric restricts fibrotic foreign body responses. After maturation, functional cells comprised 3% of the total cell mass. MO macrophage, T T cell, NK Natural Killer cell.
While most clinical experience is associated with transplant of cadaveric islets to the portal vein in the liver, additional subcutaneous, omental, and intramuscular sites have been extensively studied in preclinical models28. These sites may pose additional challenges for islet survival since the limited clinical data available suggests that unencapsulated extrahepatic transplants do not perform well29. However, encapsulated hPSC-derived PPs transplanted subcutaneously differentiate into tissue that contains functional glucose responsive cells within 4-6 months in animal models30,31. Building on this experience, two parallel first-in-human studies aimed to optimize the cell dose and perforation configuration of PEC-Direct subcutaneous implants in small numbers of T1D recipients (n=1732; n=1533) demonstrated that C-peptide, a marker produced by insulin-secreting cells, could be newly detected in some individuals at 6 months post-transplant and could persist until 24 months. A subset of patients achieved >30 pM C-peptide after meal stimulation (6/24, note some individuals were analyzed in both studies), a level that is associated with reduced T1D complications34. However, none of the individuals reached the 200 pM threshold associated with improved metabolic control34 or the 1000 pM level associated with insulin independence in cadaveric islet recipients35. For reference, postprandial C-peptide levels range from 1000-3000 pM in healthy individuals36. Importantly, the observed insulin production could be directly attributed to the VC-02 devices, and not the recovery of the recipients own cell function, since removal of the explants eliminated the improvements in C-peptide levels in two patients where this was carefully explored33. While comparison of transplanted PEC cells with cadaveric islets in terms of islet equivalents can only be approximated, these pilots delivered at most one-half the transplant volume required for insulin independence. Since the recovered devices contained mostly glucagon+ cells (16%) and only a small fraction of insulin+ cells (3%) it is not surprising that the transplants were not sufficient to improve secondary measures of glycemia. Regardless, these first-in-human studies demonstrated the overall safety of the approach in high risk (hypoglycemia unaware) patients with all serious adverse events attributed to the immunosuppressive regimen or surgical procedure, suggesting that maximizing transplant size and -cell composition were going to be crucial for clinical impact.
In an interim report of 1-year outcomes, Keymeulen et al., now provide evidence that hPSC transplants are on the cusp of providing benefit to many patients. Using an adaptive trial design, the transplant volume was increased 2-3 fold and all devices used the perforation pattern and density associated with the best outcomes in previous trials32,33 The transplant recipients were selected using similar criteria to the previous trials, requiring stable T1D (>5 years), a high risk for hypoglycemic complications (Clarke score 4), and meal-stimulated C-peptide levels 30 pM prior to transplant. With the increased dose and optimized device configuration, 3/10 recipients produced 100 pM postprandial C-peptide from 6-months post-transplant and one surpassed the 200 pM threshold associated with metabolic significance. Excitingly, this individual achieved improved time spent in the target blood sugar range (by continuous glucose monitoring), a clinically meaningful measure of function.
Now that hPSC-derived cells have been shown to produce metabolically significant amounts of insulin in a T1D patient, there is a path to match and potentially exceed the outcomes observed with cadaveric transplants. Assuming a linear relationship between -cell mass and insulin secretion, it appears that a further ~10-fold increase in functional -cell mass would be sufficient to achieve insulin-independence (>1000 pM C-peptide) in some patients and a metabolic benefit (>200 pM C-peptide) in most recipients. Unfortunately, simply further increasing the transplant size would likely increase surgical complications. Consistent across the clinical trials, recovered PEC-Direct devices contained large acellular regions filled with extracellular matrix. This material permanently occupies space that could be better utilized as cells currently comprise at most ~3% of the total volume within a device1. Although histological analysis of the PEC-Direct devices retrieved from the non-responders was not available in the interim report, further insight into the fate of transplanted PPs and the composition of infiltrating cells in failed grafts will help focus future efforts. Interestingly, in samples from two responders, the less functional graft was already dominated by infiltrating recipient cells at 3 months post-transplant and the cell mass was negligible at 9 months despite having a larger total cell volume1. Human islets are composed of ~50% cells that are interspersed with other endocrine cell types and aligned to the vasculature37. Thus, if the majority of the device volume were filled with islet-like structures, there should be a sufficient functional cell mass for most patients.
Recapitulating embryonic pancreatic development in vitro has produced PPs that clearly have the potential to complete differentiation into functional cells in a process that takes 4-6 months post-transplant. Additional clues from developmental biology indicate that there are stage-specific interactions between endogenous endocrine precursors and the vasculature that influence pancreatic differentiation. Initially, endothelial cells induce the differentiation of endocrine cells38, which then signal back to increase the density of the local vascular network39 and deposition of a vascular basement membrane that promotes cell function23. Thus, cells participate in the construction of a specialized niche through interactions with the vasculature that are essential for subsequent cell maturation. While the perforated design of the PEC-Direct device allows infiltration of endothelial cells, the growth of this vascular network takes time and is competing with recipient fibroblasts which are only partially blocked by the outer non-woven fabric layer (Fig. 1), suggesting that there are limitations to mechanical control of these processes. The strengths and weaknesses of the PEC-Direct device compared to other cell-based therapies are summarized in Table 1. Here, we highlight recent advances that could help maximize the yield of vascularized cells and in the best-case scenario provide an immune privileged niche that would eliminate the need for systemic immunosuppression (Fig. 2).
Immunomodulatory materials and cells could be used to create an immune privileged niche for transplanted PECs and further discourage fibroblast infiltration. cell numbers could potentially be increased by improving the microenvironment and converting other pancreatic cell types to the cell fate. MO macrophage, T T cell, NK Natural Killer cell. Treg Regulatory T cell, M2 M2 macrophage, CXCL12 CXCL12 chemokine.
The materials in the PEC-Direct device, particularly the outermost non-woven fabric layer, suppress a full-blown foreign body response associated with the recruitment of macrophages and fibroblasts to the interface with recipient tissues27. Limiting residual fibroblast infiltration1 might be most important in the acute post-transplant period, as they likely interfere with PP differentiation and the establishment of the intra-device vascular network. The precise composition of the perforated VC-02 encapsulation membrane remains proprietary. However, if it is composed of alginate or similar material, then biomodulatory factors could be directly integrated into the encapsulation membrane40. Notably, incorporation of the CXCL12 chemokine was recently shown to protect microencapsulated xenogeneic islets in a non-human primate model41. The primary mechanism of acute islet protection is associated with repulsion of islet-reactive effector T cells42. However, CXCL12 has multiple immune modulatory roles43, and protected islets also show reduced macrophage and fibroblast surface infiltration and collagen deposition40,41. These studies suggest that incorporating chemokine(s) such as CXCL12 into the encapsulation membrane, or potentially adding an additional biomodulatory layer, could improve the microenvironment within the device. Additional advances in biomaterials functionalized with diverse immunomodulatory molecules have been recently reviewed in the context of islet transplantation44.
Giving the vasculature a head start could be a complementary way to limit the opportunities for intra-device fibrosis. Instead of relying exclusively on the recipients vasculature, the addition of ready-made microvessels isolated from adipose tissue to hPSC-derived PPs improved early graft survival and reduced the time required for cell differentiation to less than 10 weeks in mouse T1D models45. Harvesting recipient microvessels would add additional complexity to a clinical transplant program but a proof-concept pilot study using healthy donor microvessels could be informative. Ideally, microvessel-equivalents would also be produced from hPSCs46, although scale up under GMP conditions as was done for PPs18 would also be needed.
Improving the intradevice microenvironment might increase not only the mature pancreatic cell volume within a device but potentially also the proportion of cells. In the small number of recovered grafts that have been analyzed histologically, cells comprise at most ~3% of the total cell volume1,33,34. In contrast, preclinical studies with similar device-encapsulated PPs have produced grafts with up to 16% cells by transplanting into a preformed pouch at the surgical site47. Presumably, the 5 weeks between pouch formation and device engraftment allowed for vascularization of the transplant site and resolution of acute inflammatory responses. Importantly, these data indicate that partially differentiated PPs are capable of producing significantly more cells within an optimized microenvironment. Beyond improving the host environment, an attractive source of additional cells is from transdifferentiated cells, which are invariably the most abundant pancreatic cell type identified after in vivo maturation of PPs1,47. While paracrine signals from cells are important for optimal cell function48, these intraislet interactions are unlikely to be compromised by the transdifferentiation of excess cells that are currently produced in superphysiological proportions. Furthermore, reducing cell content in the graft could have metabolic benefits as there is growing evidence that hyperglucagonemia interferes with cell function49. cells have an innate ability to transdifferentiate, although it is only triggered by near complete cell destruction50,51. Overexpression of the key cell transcription factors PDX1 and MAFA in adult cells produces -like cells with the ability to sense glucose and secrete insulin52,53, although these cells retain aspects of their previous cell identity. To avoid perturbing differentiation to the PP stage in vitro, implementing directed transdifferentiation in hPSC-derived transplants will require engineered stem cells with the ability to induce cell factors specifically in mature endocrine cells54. A further 2-3 fold increase of the cell mass observed in preclinical studies via transdifferentiation would produce structures with very similar cellular composition to endogenous human islets37.
The ultimate goal of a hPSC-based therapy for T1D is to provide long-term cell function without the need for systemic immunosuppression. Cotransplantation of microgels containing individual immunomodulatory factors such as PD-L155 or FasL56 can have profound effects on graft survival in immunocompetent hosts. For example, FasL presenting microbeads combined with only two weeks of rapamycin monotherapy supported graft function for over six months and induced Treg-dependent local tolerance without systemic effects on the immune system in an allogeneic mouse model56. Similar effects were seen in non-human primates57, although the long-term viability of the grafts was not evaluated.
In addition to achieving allogeneic graft tolerance, hPSC-derived cells must contend with the dysregulated autoimmune response in T1D. Excitingly, it now appears possible to fully cloak hPSCs and their differentiated progeny by overexpressing a cocktail of 8 immunomodulatory factors that includes PD-L1 and FASL58. Together, these factors disrupt antigen presentation, T-cell and NK cell attack, and innate inflammatory responses. By activating a proliferation-dependent kill switch59, cloaked cells could be maintained in a dormant state within an immunocompetent host. Furthermore, these cloaked cells protected their neighbors, including allogeneic islets and xenogeneic hPSCs. While PPs could potentially be generated directly from cloaked hPSCs, the overexpression of 8 genes might impact cell function long-term. An elegant strategy to address all the key issues discussed here would be to generate cloaked endothelial cells for cotransplantation with PPs that are genetically primed for to cell transdifferentiation.
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Finishing the odyssey to a stem cell cure for type 1 diabetes - Nature.com
- Collaborative Research on Innovative Cartilage Organoid Cell Therapy for Intervertebral Disc Degenerative Disease using Astellas' Universal Donor Cell Technology -
TOKYOand OSAKA, Japan, July 22, 2024 /PRNewswire/ -- Astellas Pharma Inc. (TSE: 4503,President and CEO: Naoki Okamura,"Astellas") and Graduate School of Medicine / Faculty of Medicine, Osaka University (President: Shojiro Nishio"Osaka University") today announced that Astellas Institute for Regenerative Medicine (a wholly owned subsidiary of Astellas, "AIRM"), Universal Cells (a wholly owned subsidiary of Astellas) and Osaka University have entered into a research collaboration to develop innovative pluripotent stemcell*1-derived cartilage organoid cell therapy for the treatment of intervertebral disc degenerative disease*2.
Universal Cells holds the rights to Universal Donor Cell (UDC) technology to create cell therapy products from pluripotent stem cells that have reduced risk of immune rejection by genetically modifying Human Leukocyte Antigen (HLA) using gene editing technology.
Under the terms of the agreement, the three parties aim to combine the cartilage tissue creation protocol established by Professor Noriyuki Tsumaki of (Graduate School of Frontier Biosciences / Premium Research Institute for Human Metaverse Medicine) the Department of Tissue Biochemistry and Molecular Biology, Graduate School of Medicine, Osaka University, a leading researcher in cartilage diseases, Universal Cells' UDC technology, and AIRM's exceptional R&D expertise in cell therapy, and jointly create an innovative cell therapy for intervertebral disc degenerative disease.
Yoshitsugu Shitaka, Ph.D., Chief Scientific Officer (CScO) of Astellas"Astellas is committed to achieving our VISION of being "on the forefront of healthcarechange, turning innovative science into VALUE for patients". We hope to provide our cutting-edge UDC technology to academia and startups globally, and deliver next-generation cell therapies to patients. This partnership is an important step in the open innovation using UDC technology."
Professor Noriyuki Tsumaki, M.D., Ph.D., (Graduate School of Frontier Biosciences / Premium Research Institute for Human Metaverse Medicine) Department of Tissue Biochemistry and Molecular Biology, Graduate School of Medicine, Osaka University"We believe that our cartilage-like tissue has the potential to regenerate intervertebral discs. We hope that combining our research with Astellas' UDC technology and R&D cell therapy system will accelerate and realize the development of regenerative therapies to treat intervertebral disc degenerative disease."
*1 Pluripotent stem cell: Cells that possess the ability to proliferate almost indefinitely and differentiate into any cell that makes up the organism. Ex. embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). *2 Intervertebral disc degenerative disease: A type of degenerative spinal disease. The intervertebral discs, which are cartilaginous tissues, play a crucial role as cushions between each bone of the spine by containing a significant amount of water. This helps maintain flexible movement of the back. However, when these discs degenerate, they lose water, resulting in the failure of their cushioning function, which can lead to lower back pain.
About AstellasAstellas Pharma Inc. is a pharmaceutical company conducting business in more than 70 countries around the world. We are promoting the Focus Area Approach that is designed to identify opportunities for the continuous creation of new drugs to address diseases with high unmet medical needs by focusing on Biology and Modality. Furthermore, we are also looking beyond our foundational Rx focus to create Rx+ healthcare solutions that combine our expertise and knowledge with cutting-edge technology in different fields of external partners. Through these efforts, Astellas stands on the forefront of healthcare change to turn innovative science into VALUE for patients. For more information, please visit our website at https://www.astellas.com/en.
Cautionary Notes(Astellas)In this press release, statements made with respect to current plans, estimates, strategies and beliefs and other statements that are not historical facts are forward-looking statements about the future performance of Astellas. These statements are based on management's current assumptions and beliefs in light of the information currently available to it and involve known and unknown risks and uncertainties. A number of factors could cause actual results to differ materially from those discussed in the forward-looking statements. Such factors include, but are not limited to: (i) changes in general economic conditions and in laws and regulations, relating to pharmaceutical markets, (ii) currency exchange rate fluctuations, (iii) delays in new product launches, (iv) the inability of Astellas to market existing and new products effectively, (v) the inability of Astellas to continue to effectively research and develop products accepted by customers in highly competitive markets, and (vi) infringements of Astellas' intellectual property rights by third parties. Information about pharmaceutical products (including products currently in development) which is included in this press release is not intended to constitute an advertisement or medical advice.
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Astellas and Graduate School of Medicine / Faculty of Medicine, Osaka University Enter into Research Collaboration to Develop Pluripotent Stem...
Brain organoids are 3D, lab-grown models designed to mimic the human brain. Scientists normally grow them from stem cells, coaxing them into forming a brain-like structure. In the past decade, they have become increasingly sophisticated and can now replicate multiple types of brain cells, which can communicate with one another.
This has led some scientists to question whether brain organoids could ever achieve consciousness. Kenneth Kosik, a neuroscientist at the University of California, Santa Barbara, recently explored that possibility in a perspective article. Live Science spoke with Kosik about how brain organoids are made, how similar they are to human brains and why he believes that brain organoid consciousness is not likely anytime soon.
Related: In a 1st, 'minibrains' grown from fetal brain tissue
EC: What are brain organoids, and how do scientists make them?
Kenneth Kosik: A brain organoid is made from stem cells. You can take any person and convert their, say, skin fibroblasts into stem cells, and then differentiate them into neurons. It's what stem cells are all about stem cells are called "pluripotent" because they can make any cell in the body.
We spent a fair amount of time before organoid technology came along, taking human-induced pluripotent stem cells and inducing them in a two-dimensional array to look at neuronal differentiation.
So that takes us halfway there. But it only gets us as far as two dimensions. And then the big insight, which came from Yoshiki Sasai in Japan and Madeline Lancaster, was to take these neurons that were beginning to differentiate cells relatively early in development and put them in a drop of what's called Matrigel a gel that can be either a liquid or a solid depending on the temperature.
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So the cells are in this drop, and then the magic happens. Instead of growing in two dimensions, they start to grow in three dimensions. It absolutely fascinates me that when biology begins to explore the third dimension, a very novel biology emerges. Certainly, in two dimensions, these neurons that were growing could achieve a very wide diversity of cell types, but they did not achieve any kind of interesting anatomy.
Once they're growing in three dimensions, they start to form relationships to each other, kinds of structure and anatomy, that has a very loose resemblance to the brain. And I really emphasize the word "loose," because there are people that use a misnomer for brain organoids and call them "minibrains." They're not brains at all. They are organoids meaning like the brain.
A question we're keenly interested in, and many labs are, is that if organoids are like the brain, to what degree do they resemble the brain and to what degree do they differ? And they differ from the brain a lot, so you have to be very careful about interpretations of organoids. Not everybody thinks that organoids are going to be informative for neuroscience because what we find in an organoid may be over-interpretation. But on the other hand, [it] is forming a three-dimensional structure that has some degree of lamination [formation of layers of cells within tissue], it has these rosettes in which, from the center of the rosette, you can progressively see cells becoming more mature as development proceeds, which is very similar to what happens in the brain.
Related: Lab-grown 'minibrains' may have just confirmed a leading theory about autism
EC: Are there any brain organoids that accurately capture the whole brain yet?
KK: There is no organoid that captures the whole brain. There are approaches that attempt to capture more of the brain than, say, just the one part that maybe we and other labs are working on. These are called "assembloids." [Scientists] take stem cells and differentiate them down a pathway that may make a little more ventral [front part of the] brain, or a little more dorsal [back part of the] brain, and they put them together, they fuse them, so that you get more comprehensive fusion a wider representation, I should say, of brain anatomy.
There are other ways of making organoids that are a little more indiscriminate. They're not directing the stem cells towards dorsal and ventral, they are putting them all together. That's a lot of what we do. Those were the techniques that were originated by Lancaster. And in that case, it's my opinion that when you do it that way, you get a broader representation of cell types. That's what you gain, but you sacrifice anatomical accuracy because when you make an assembloid, the anatomy is not great. But when you do it without differentiating toward dorsal and ventral and you put it all together, the anatomy becomes even more problematic.
EC: As you alluded to, these organoids are similar to human brains, but there's some key physiological differences. Can you explain those?
KK: So, one similarity right away is that you see a lot of spiking going on.
(Editor's note: Kosik is referring to the fact that, when an organoid is hooked up to electrodes, this triggers electrical spikes, or signals, transmitted between neurons.)
It's quite remarkable, and underlying this is the notion, which is probably what intrigues me the most, that all of this activity is spontaneous: it just arises based on the assembly of the neurons.
And now we can look at the relationships of those spikes. When you do that, you can ask the question, well, if I see neuron A firing, what's the likelihood that I'll see neuron B firing? I'm going to look at the binary relationships among all of them and I'm doing it with the filter that when neuron A fires I'm only going to look at when another neuron fires within 5 milliseconds. Why 5 milliseconds? Because that's about the time in which it takes for transmission to occur across the synapse. (Editor's note: A synapse is the gap between two neurons.)
And when we do that, you can see that they form a network. You connect A and B, and then you connect C and D, and then A and C. You can see that the neurons are talking to each other and this arises spontaneously.
That is one example of the way in which an organoid does something that will spontaneously resemble what happens in the brain.
The way I look at an organoid, it is a vehicle that has the capacity to encode experience and information if that experience were available to it but it's not. It has no eyes, ears, nose or mouth nothing's coming in. But the insight here is that the organoid can set up spontaneous organization of its neurons so that it has the capacity to encode information, when and if it becomes available. That's just a hypothesis.
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EC: Do you think that brain organoids will ever achieve consciousness?
KK: So that's where things get a little mysterious. I think that those kinds of questions are predicated on this term that people have a lot of trouble defining: consciousness.
[Based on currently fashionable theories of consciousness] I would say, "No, it doesn't even come close."
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EC: You spoke about the fact that organoids have shown some capacity to encode information, but they don't have the experience to do this in the first place. What would happen if, hypothetically, a human brain organoid was transplanted into an animal? Could it then achieve consciousness?
KK: Let's break that down. Before it is transplanted into an animal, some people would say the animal already has consciousness and some people would say [it does] not. So, right away, we get into this difficulty about where in the animal kingdom does consciousness begin? So, let's reframe the question. If you then took an animal, which may or may not have some degree of consciousness, and you transplant in a human organoid, would you confer consciousness on that animal or would you enhance consciousness, or would you even get something that resembled human consciousness in the animal? I don't know the answer to any of those questions.
We can do these hybrids now so it's a good question. But the evaluation of consciousness now, because of all the problems as to what consciousness is, is still going to be an open question.
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EC: Do we have an idea of rough timescales , is consciousness something that could happen in the near future, after, say, a certain number of years, or is it still really uncertain at this point?
KK: Technology is moving very fast. One place where we may begin to push the boundary is in the so-called cyborgs, or organoid interfaces. That would be one direction that could be interesting. Maybe a little bit toward consciousness, but even more so toward developing the implementation of human abilities in one of these synthetic systems.
EC: Can you think of any obvious benefits and drawbacks of these organoids being able to achieve consciousness?
KK: We know so little about neuropsychiatric conditions. Neuropsychiatric drugs are developed without understanding any deep physiology. All of that could be done, I think, with organoids. I think as disease models, it could be very, very useful [for them to achieve consciousness].
The dream state that I have is to develop them as computational systems because, right now, to do the kinds of very expensive computations that are required for ChatGPT and many of these large language models, these take hundreds of millions of dollars to develop. They require a server farm of energy to keep them going. We're really just running out of computer power. Yet, the brain does a lot of this stuff on 20 watts. So, a big interest for me is, "Can organoids, if not solve, contribute to the huge demands that we're making on the energy system by tapping into the highly efficient way in which the brain, and presumably the organoid, can handle information?"
Editor's note: This interview has been edited and condensed.
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'We can't answer these questions': Neuroscientist Kenneth Kosik on whether lab-grown brains will achieve consciousness - Livescience.com
There have been several studies looking at how MS patients respond to stem cell transplantation, a method where patients are infused with healthy stem cells in hopes of resetting their immune system.
Past research has shown remyelinating and immunomodulatory functions representing a potential therapeutic option for MS patients.
Now, new research, published in Nature Scientific Reports on May 31, revealed that people with MS are more prone to experience a short-term reduction in disability and brain lesion volume after receivingstem cell therapy.
The study, conducted by faculty at Zagazig University in Zagazig, Egypt, was led by Asmaa Ahmed Nawar, and involved a meta-analysis of nine studies detailing randomized clinical trials.
From a literature search of 3,948 records, the research team looked at randomized control trials of stem cell therapy in MS patients in 422 patients collected from PubMed, Web of Science, Scopus and Cochrane Library.
We prepared this study following the PRISMA checklist and performed all the steps according to the Cochrane Handbook for Systematic Reviews of Interventions, Nawar explained. We included cross-over trials to increase the sample size of the analysis to get credible results, and these studies were included until the cross-over point to avoid the carry-over effect in such trials.
From the data, it was determined that stem cell therapy significantly improved MS patients expanded disability status scale following twomonths, and reduced brain lesion volume during the first two months as well. Therefore, the team concluded stem cell therapy does improve the disability of MS patients and reduce their brain lesion volume. The research team further found that stem cell therapy was safe, with zero cases of mortality during the follow-up period.
An interesting finding was that those who received stem cell therapy showed clinical improvements for those patients who received their own hematopoietic stem cells as opposed to those who received mesenchymal stem cells, proving the value of the former.
Despite the positive findings of clinical improvement in the early months, the researchers discovered that after 12 months, there were no differences in disability between patients who underwent stem cell therapy and controls, or individuals with MS who received another treatment or a placebo.
Additionally, the authors noted that those who underwent stem cell therapy showed not significant improvement in motor function, hand dexterity or cognitive function.
Therefore, Nawar and his research team suggested further study of stem cell therapy in MS patients was important, noting in the paper that longer follow-up can help to detect the long-term effect on disease progression and determine any long-term safety concerns. The team also encourages the research of different types of stem cell therapy to better find those that result in optimal results.
Continue reading here:
New Study Shows Short-Term Benefits of Stem Cell Therapy for MS Patients, But Long-Term Efficacy Remains Unclear - Managed Healthcare Executive