Category Archives: Stem Cells

BORDEAUX, France, July 9, 2024 /PRNewswire/ -- TreeFrog Therapeutics, the French biotech company advancing a pipeline of regenerative medicine cell therapies based on a disruptive proprietary technology platform will present at several events at the global stem cell event in Hamburg, including a poster session with data from their lead program in Parkinson's Disease and an innovation showcase with a deep dive on their transformational technology, C-Stem.

Regenerative medicine holds immense potential in addressing some of the largest unmet needs in diseases of the major organs such as the central nervous system, the liver, pancreas and heart. However, despite major advances in the last 70 years, there are still bottlenecks holding up innovation, particularly, the ability to produce the required amount of high-quality cells, efficiently.

TreeFrog's lead program in Parkinson's disease has proven efficacy in pre-clinical models using a unique approach of grafting 3D format microtissues containing dopaminergic progenitors and mature dopaminergic neurons, as opposed to single-cell suspensions. The poster, presented by Maxime Feyeux, Chief Scientific Officer and Co-founder of TreeFrog will highlight the therapeutic potential of the 3D approach, and delve deeper into the characterization of the product through complementary methods including qPCR, RNAseq, flow cytometry and microscopy.

The pipeline of TreeFrog is based on their C-Stemtechnology, the culmination of over 20 years of research bringing the best in biophysics and stem cell biology together. This breakthrough technology addresses the challenges of scale and quality, and the closed system enables both the amplification and differentiation of cells. Maxime will be joined by two senior scientists Joffrey Mianne, Head of iPSC Research and Clement Rieu, Head of Technologies R&D - during an innovation showcase about C-Stem.

Presentations details:Poster Presentation: #190 Clinical Applications (CA) SessionWednesday, July 10, 2024 @ 6:45 PM 7:45 PM Room: Poster & Exhibit hall

Innovation Showcase Thursday July 11th, 2024 @ 6:00PM 6:30PM Room: Hall 3, entrance level

AboutTreeFrog TherapeuticsTreeFrog Therapeutics is a biotech company set to unlock access to cell therapies for millions of patients bringing together biophysicists, cell biologists and bioproduction engineers to address the challenges of producing and differentiating cells of quality at unprecedented scale, cost-effectively. To realize their mission of Cell Therapy for all, TreeFrog has their own therapeutic programs and partnerships with leading biotech and industry players in other areas.

http://www.treefrog.fr

Contact: Rachel Mooney Chief Communications Officer TreeFrog Therapeutics [emailprotected]

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SOURCE TreeFrog Therapeutics

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TREEFROG THERAPEUTICS PARTICIPATES IN AN INNOVATION SHOWCASE & POSTER SESSION AT THE INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH (ISSCR) ANNUAL...

Our teeth cant repair themselvesbut what if they could? The future of dentistry lies in the captivating field of regenerative medicine, where stem cell research is diving deep into the potential to repair damaged teeth with living fillings. But how far are we from ditching fillings for specialized tooth restoration? While the research is science fact, getting a living filling from your dentist is still science fictionfor now.

I find this field really fascinating, says New York cosmetic dentist Victoria Veytsman, DDS. The field of tissue engineering and regenerative medicine in dentistry is really at the forefront of where healthcare is going.

Stem cells are those super useful specialized cells (found in adult body tissues and in embryos) that can be guided towards becoming many different cell types and can self-replicate. That makes them immensely useful in regenerative medicine, where the goal is to get the bodys repair processes engaged to handle damaged, diseased or otherwise unwell tissues. According to the California Institute for Regenerative Medicine, the most commonly used stem cell-based therapy is for bone marrow transplants.

When it comes to filling a cavity with them, stem cells alone arent enough to complete the process of tooth restoration, explains Dallas, TX cosmetic dentist Salvator La Mastra, DDS. They would need a framework of some kind in order to form in the correct manner.

Dr. Veytsman explains that current research is focused on creating that framework, creating a kind of living filling.

We dont want enamel to grow in a petri dish; we want it to grow on your tooth, Dr. Veytsman says. So the process requires a scaffold or matrix to support that growth.

When a tooth develops a cavity, the first step is to remove the decay and stop the process of damage. Cavities are caused by bacteria, Dr. La Mastra explains. That acid producing bacteria is what causes the cavitation of the tooth, which is the cavity itself and the decay. Its basically necrotic tissue that we have to drill out.

Then, you have to fill in whats lost. We do things like crowns and fillings to replace the chief structure that was lost or decayed, Dr. Veytsman explains. Its called restorative dentistry because were trying to restore whats been lost.

Those fillings are made of amalgam (a mixture of metals) or composite resin filling materials (made from polymers and glass particles), and we know theyre safe, functional and that they wont decay in the future. Thats something we cant say about these living fillings.

One thing about our current implants and fillings is that we know they wont develop cavities down the line, La Mastra says. There are complications that could arise from the regenerative method that could cause more than just aesthetic consequences; your bite can also be impacted.

I think were just at the beginning of this technology, Dr. Veytsman says. But it definitely has the potential to change the way we approach cavities in the years to come.

Stem cells could also be utilized outside of living fillings to benefit oral health. Aside from repairing enamel, stem cells could be used to encourage the growth of dentin, restore pulp, even regenerate lost gum tissues.

Youre seeing the rise of stem cell banking now for these purposes, Dr. Veytsman explains. Harvesting and banking stem cells for future applications and to use as a preventative measure are growing in popularity.

I think were multiple decades away from a changeover to regenerative medicine in dentistry, La Mastra says. I already have patients who ask me if they can just regrow their tooth, and we are nowhere near being able to do that.

While living fillings arent going to enter your dentists office in the immediate future, theres still reason to be excited.

The advent of AI technologies is really accelerating this research, Dr. Veytsman says. And its letting us ask a ton of questions about possible applications. Can regenerative medicine deal with prevention? Can it help stop decay in the very early stages? Were still so early in this process, but AI and regenerative medicine are really at the forefront of healthcare right now.

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"Living Fillings" Could be the Future Thanks to Stem Cells - NewBeauty Magazine

UC San Diego scientists have found the most common form of liver cancer could be targeted and treated more effectively using a stem cell-derived therapy, according to a report published Tuesday.

The report, published in the scientific journal Cell Stem Cell, focuses on hepatocellular carcinoma, a cancer with a high mortality rate.

While not yet studied in patients, the treatment which involves the lab engineering of natural killer white blood cells to battle HCC could be mass-produced and ready for deployment rapidly, the researchers said.

Unlike a treatment called chimeric antigen receptor-expressing T-cell therapy, which requires patient personalization, the NK-cell therapy could be more acceptable to more bodies.

To some extent all tumor cells perhaps hepatocellular carcinoma more so inhibit immune cells that try to kill them, said UCSD School of Medicine Professor Dr. Dan Kaufman, lead author on the study and director of the Sanford Advanced Therapy Center at the universitys Sanford Stem Cell Institute and Moores Cancer Center member.

This is one key reason why some immunotherapies like CAR T cells have been less successful for solid tumors than for blood cancers the immunosuppressive tumor microenvironment.

Kaufman and his team produced the NK cells in which a protein that impairs immune function was disabled. Hepatocellular carcinoma tumors and the liver in general contain large amounts of the substance, which both inhibits the immune cell activity and allows cancer to proliferate, the authors write.

Typical NK cells without the disabled receptor, like CAR T cells, were not very effective in battling the cancer.

These are pretty resistant tumors when we put them in mice, they grow and kill the mice, Kaufman said.

The five-year survival rate for HCC in humans is less than 20% and is responsible for more than 12,000 deaths in the United States annually.

However, when researchers tested the modified NK cells against the cancer, we got very good anti-tumor activity and significantly prolonged survival.

These studies demonstrate that it is crucial to block transforming growth factor beta at least for NK cells, but I also think its true for CAR T cells, Kaufman said. If you unleash NK cells by blocking this inhibitory pathway, they should kill cancer quite nicely.

He said his teams discovery will likely be reflected in clinical trials for many groups working with the various therapies or solid tumors.

City News Service, Inc.

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UCSD Team Tests Stem Cell-Derived Therapy On Liver Cancer; Results Promising - Times of San Diego

CAMBRIDGE, Mass., July 9, 2024 /PRNewswire/ --Factor Bioscience Inc., a Cambridge-based biotechnology company focused on developing mRNA and cell-engineering technologies,announced its participation in the International Society for Stem Cell Research (ISSCR) 2024 Annual Meeting to be held in Hamburg, Germany from July 10-13, 2024. Factor will deliver six presentations covering the latest preclinical data from Factor's cell engineering platforms.

"We are excited to showcase our recent progress on developing next-generation therapies based on cutting-edge stem cell science at ISSCR 2024," said Dr. Matt Angel, Co-Founder, Chairman and CEO of Factor. "The work that we will be presenting this week represents more than a decade of focused effort. We are committed to developing these new medicines to enable a brighter future for patients and their families."

The work that we will be presenting this week represents more than a decade of focused effort.

Dr. Kyle Garland, Factor's Director of Translational Science, added, "Our six presentations at ISSCR 2024 will cover several novel and unique stem cell technologies, including iPSC-derived macrophages engineered with mRNA to enhance T cell cytotoxicity to solid tumor cells. We are excited to share these and other advances in Hamburg over the next few days."

Details of the presentations are below:

For more information about the International Society for Stem Cell Research (ISSCR) 2024 Annual Meeting, visit http://www.isscr2024.org.

About Factor BioscienceFounded in 2011, Factor Bioscience engineers cells to promote health and improve lives. Factor Bioscience is privately held and headquartered in Cambridge, MA. For more information, visit http://www.factorbio.com.

SOURCE Factor Bioscience

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Factor Bioscience to Deliver Six Presentations at the International Society for Stem Cell Research (ISSCR) 2024 Annual Meeting - PR Newswire

Conor McGregor has finally broken his silence regarding the injury that led him to pull out from UFC 303. UFCs rival promotion Bellator returned to Ireland for another event, and surprisingly The Notorious was there in attendance. As reporters caught hold of McGregor, he revealed the experience hes going through as he recovers from his injury.

When the UFC announced that Conor McGregor had gotten injured, a cloud of ambiguity surrounded the situation because they wouldnt reveal what the injury was. But the Irishman would come out days later to reveal that he had his toe broken, and as far as his recent statements are concerned, it has not been a great time for McGregor and he had to go through a special treatment for it.

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The first thing that most people want to know is when Conor McGregor will be in action, and guess what? The Irishman has certainly made up his mind on when he wants to fight Michael Chandler. McGregor wants to make his return in the closing stages of the summer season. He would require some recovery time because hes still nursing his toe injury carefully.

August or September Id love [to fight Michael Chandler] Im in recovery mode. Three weeks to the day. It happened three weeks ago today and Im still here in the slippers, said Conor McGregor during an interview after the Bellator Dublin event, where he came to support his teammate, Sinead Kavanagh, and even cornered her. The injury, according to the Irishman, is so severe that he cannot even get his foot to go inside a shoe.

Theyre [the slippers] cozy enough but I cant get into a shoe yet. So you know they put stem cell into me for the camp, took it from the back, they put it in me foot. 20 milligrams of stem cells, Conor McGregor added. And for that, he went through a special kind of treatment- stem cell therapy.

via Imago

LAS VEGAS, NEVADA JULY 10: Conor McGregor of Ireland prepares to fight Dustin Poirier during the UFC 264 event at T-Mobile Arena on July 10, 2021 in Las Vegas, Nevada. (Photo by Chris Unger/Zuffa LLC)

Stem cell therapy has proved to be quite the game-changer as many athletes prefer it. It helps the body garner the ability to regenerate and even repair damaged tissues, or tissues which are affected due to certain kinds of diseases. When Conor McGregor had his treatment, the doctors took stem cells from his back and added those to his toe region.

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Moreover, he would go on to make a shocking revelation and claim that his toe injury was way more painful than getting his leg broken almost three years ago.

The Notorious, during that interview, would also claim that hes dialed infor the Michael Chandler fight. However, the foot injury hes dealing with isnt something thats given him a moment of peace because its been a pretty painful experience. And guess what? McGregor claims that his current injury is way worse than the leg break he suffered against Dustin Poirier.

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Im good. The injury is, its a nuisance. Its painful. Im not gonna lie. Its very, very painful. Probably more painful than the leg and thats the truth, said Conor McGregor. Now, the question that arises with this statement is why would he make such a comparison with his leg break because that injury, for everyone, seemed like a career-ender. Well, the reason behind his claims is that his toe gets more exposure than his leg. Nevertheless, McGregor understands that injuries are part and parcel of combat sports. The leg, at least, was wrapped up. I couldnt access it. The foot is just there still Its exposed or something. So, its a bit painful but it comes with the f***ing territory, Conor McGregor added.

Having heard what Conor McGregor had to say about his injury, its unlikely that Dana White would come out to talk about his return, which he only affirmed during the post-fight pressers at UFC Saudi Arabia. Hence, it remains to be seen how long will he take to get fight-ready. What are your thoughts on this situation? Drop your comments below.

Excerpt from:
Conor McGregor Gives Official Status on Return With Shocking Disclosure Behind Injury They Put Stem Cell Into Me ... - EssentiallySports

PCs as the origin of intestinal tumors in the context of inflammation

We first reported on the ability of PCs to re-enter the cell cycle and dedifferentiate upon irradiation and inflammation to acquire stem cell-like features and contribute to the tissue regenerative response11,12,13. Consequently, we questioned whether PCs could be the origin of intestinal cancer in the context of inflammation. We bred mice carrying lox alleles at the tumor suppressors and oncogenes most frequently mutated along the adenoma-to-carcinoma sequence, namely Apc14, Kras15 and Trp53 (encoding p53)16, each combined with Cre specific for Lgr5+ ISCs (Lgr5CreERT2-EGFP)17 or for PCs (Lyz1CreERT2)18. Following Cre activation by tamoxifen, dextran sulfate sodium (DSS) was administered through drinking water to model inflammation (Fig. 1a). In the absence of DSS-induced inflammation, PC-specific single gene mutations did not give rise to intestinal tumors. By contrast, loss of Apc in Lgr5+ ISCs transformed crypts into -cateninhi foci that grew into multiple adenomas 46weeks after Cre induction (Fig. 1b). When single gene mutations were combined with DSS administration, Apc loss in PCs resulted in increased nuclear and cytoplasmic -catenin expression eventually leading to the formation of PC-derived adenomas (Fig. 1c). Of note, Paneth-specific Kras or Trp53 mutations did not result in tumor formation even in the presence of the inflammatory stimulus (Fig. 1d). However, the compound loss of Apc and oncogenic activation of Kras in PCs resulted in a striking increase in tumor multiplicity (6.1-fold) even in the absence of DSS (6.9-fold) (Fig. 1e). The combination of Apc and Trp53 mutations in PCs also led to an increase in tumor multiplicity upon DSS administration (1.6-fold), although to a lesser extent when compared with the compound Apc/Kras-mutant genotype, possibly indicating a distinct mechanism underlying tumor onset in these mice. Indeed, phospho-histone H2A.X (Ser139) immunohistochemistry (IHC) analysis confirmed an increase in DNA damage and chromosomal instability in Trp53-mutant tumors (Extended Data Fig. 1a). Targeting all three genes in PCs resulted in a very aggressive phenotype with high tumor multiplicity (10.1-fold) in the absence of the inflammatory stimulus (Fig. 1e). When compared with Apc-driven tumors that originated in PCs, the histology of adenomas from mice in which two or three genes were targeted revealed a progressive increase in dysplasia and invasive morphology (Extended Data Fig. 1b). The distribution of adenomas along the small intestine also followed distinct patterns with a prevalence of duodenal tumors in compound Apc/Kras tumors regardless of DSS (Extended Data Fig. 2a).

a, Crelox strategy aimed at the targeting of Apc, Kras and Trp53 mutations in ISCs (Lgr5+ ISCs) and PCs (Lyz1+ PCs). w/wo indicates the presence (with) or absence (without) of DSS. b,c, -Catenin IHC analysis of intestinal tumors initiated from Lgr5+ ISCs (b) and PCs (c). Asterisks indicate Lgr5+ ISCs and PCs with enhanced cytoplasmic and nuclear -catenin accumulation; tumor foci and adenomas are indicated by dashed lines. d,e, Tumor multiplicity was calculated according to tumor-bearing mice (d) and by tumor number per genotype (e) in the presence/absence of DSS and based on Swiss roll counts. Error bars denote s.d. P values denote one-way ANOVA and Tukeys post hoc tests for group comparisons. f, Lineage-tracing analysis of PCs, labeled using yellow fluorescent protein (YFP), at different stages of tumor initiation and progression. gj, LYZ1 (g) and DCLK1 (i) IHC analysis of Lgr5+ ISC-derived (left) and PC-derived (right) adenomas, and quantification of number of Lyz1+ (h) and Dclk1+ (j) tumor cells. P values depict one-way ANOVA and Tukeys post hoc tests for group comparisons. k, Lyz1, Dclk1 and Axin2 quantitative PCR expression analysis across different adenoma genotypes. P values represent one-way ANOVA and Tukeys post hoc tests for group comparisons.

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To validate the PC origin of the observed intestinal tumors, we bred Lyz1CreERT2 mice with R26LSL-tdTomato or R26LSL-YFP reporters and traced their lineage upon tamoxifen-driven targeting of the Apc, Kras and Trp53 mutations. As shown in Fig. 1f and Extended Data Fig. 2b, this confirmed the PC origin of the corresponding tumors by capturing the process from microscopic lesions to adenoma formation.

Overall, these results demonstrate that PCs can initiate intestinal adenomas upon genetic ablation of Apc in the context of inflammation. In combination with Apc loss, activation of oncogenic Kras or loss of Trp53 function rescues the need for an inflammatory stimulus and results in increased PC-derived tumor multiplicities and progression to a malignant phenotype.

Next, we characterized lineage-specific markers in PC- and ISC-originated tumors using IHC. Of note, although cells expressing the PC marker lysozyme (LYZ1) were notable in Lgr5-derived tumors (Lgr5/Apc: 30.0%18.5% positive tumor cells), they were almost absent in adenomas that originated from PCs (Lyz1/Apc: 0.48%1.16%) (Fig. 1g,h). The opposite was observed for DCLK1 (doublecortin-like kinase 1), a Tuft19 and tumor stem cell marker20,21, that was more frequently detected among PC-derived adenomas (Lyz1/Apc: 54.1%10.5%) when compared with Lgr5-derived tumors (Lgr5/Apc: 15.6%15.7%) (Fig. 1i,j). Other lineage-specific markers for enteroendocrine (CHGcA), goblet (MUC2) and stem cells (OLFM4) showed variable levels without clear-cut differences among tumors with different cells-of-origin (Extended Data Fig. 2c). The increased Dclk1 expression in PC-derived tumors is of interest in view of its association with increased immune and stromal infiltration in colon cancer22.

To confirm these results at the transcriptional level, expression levels of Lyz1 and Dclk1 genes were analyzed by quantitative PCR with reverse transcription (Fig. 1k). Indeed, Lyz1 expression was lower in Paneth-derived tumors (Lgr5/Apc versus Lyz1/Apc: log2-transformed fold change=2.64, P=7.5104) when compared with Lgr5-derived tumors. Dclk1 expression was very low and variable at the RNA level, and did not show significant differences across the groups.

To assess the relative activation of the WNT signaling pathway among the different tumor groups, we measured expression levels of Axin2, a well-established WNT downstream target. Axin2 expression was higher in Lgr5-derived tumors compared with PC-derived tumors (Lgr5/Apc versus Lyz1/Apc: log2-transformed fold change=2.12, P=0.017) (Fig. 1k). Moreover, both Kras oncogenic activation and inflammation gradually increased Axin2 levels in PC-derived tumors, in agreement with the previously reported synergism between Apc and Kras mutations in activation of the WNT pathway23.

Thus, upon tumorigenesis, PCs dedifferentiate to a state that hampers secretory differentiation leading to specific patterns of tumor histology and gene expression distinct from that of Lgr5-derived tumors.

To elucidate the mechanisms that underlie the conversion of PCs into cells-of-origin of small intestinal tumors in the context of inflammation and/or of specific genetic hits, we combined single-cell RNA sequencing (scRNA-seq) analysis with lineage tracing. To this end, we induced the Apc, Kras and Trp53 genetic mutations in R26LSL-tdTomato/Lyz1CreERT2 (or R26LSL-YFP) reporter strains in the presence or absence of DSS (Fig. 2a). Subsequently, cells were harvested from the intestinal epithelium, purified by FACS and transcriptionally profiled by scRNA-seq (Methods and Extended Data Fig. 3). After preprocessing, we obtained the transcriptomes of 23.231 epithelial cells from 32 mice, distributed over the different lineages of the intestinal epithelium (Fig. 2b). Close examination of cells positive for the reporter genes revealed novel clusters of PCs that arise upon DSS administration and specific gene mutations, but were not observed among PCs under homeostatic conditions (PC cluster 14, Fig. 2c).

a, Schematics of the experimental approach, adapted from ref. 63, Springer Nature Limited. After genetic targeting of PCs, intestinal crypts were extracted, and the isolated cells were labeled with hashing antibodies and sorted according to three different strategies: epithelium, PC-enriched and PC-traced cells. b, UMAP embedding of the different cell clusters or lineages (left), annotated according to the expression of canonical marker genes (right). EE, enteroendocrine cells; EP, enterocytes progenitors; TA, transit-amplifying cells. c, Bar plot of the distribution of traced cells across the different mouse genotypes and experimental conditions. d, Violin plots representing marker genes of the newly identified Paneth-derived cell clusters (PC cluster 14). e, Association analysis of the RSC signature with PC cluster 14. The P value denotes the result of one-way ANOVA. f, RNA in situ hybridizations of the Clu gene in small tumors derived from PCs upon compound targeting of Apc and Kras mutations. g, Gene sets variation analysis among refs. 30,64 and the current study. EMT, epithelila-to-mesenchymal transition.

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To characterize the novel PC-derived states, we performed differential expression analysis and identified cluster-specific markers (Fig. 2d and Supplementary Table 1). Whereas PC cluster 1 appeared at low frequency across different genotypes, PC cluster 2 arises directly upon exposure to the inflammatory stimulus. Both PC cluster 1 and cluster 2 are characterized by increased expression of two markers of radio-resistant and secretory progenitors with self-renewal capacity during regeneration, namely Krt19 (ref. 24) and Atoh1 (ref. 25), whereas increased expression of Reg3b, known for its protective role in the development of colitis and ileitis26, and Cdkn1a (encoding p21), a marker of terminal differentiation in the intestine27, was observed in cluster 2 compared with cluster 1.

PC cluster 3 became apparent in mice carrying Apc mutations (7.23%5.77% of traced cells) alone and in combination with DSS treatment (16.40%2.10% of traced cells), and in double- and triple-mutant (AP, AK and AKP) mice, although not in mice carrying single Kras or Trp53 mutations. PC cluster 3 showed increased expression of Gif (gastric intrinsic factor), Cd81, a tetraspanin family member known to mark the response to gamma-irradiation and correlated with the expression of ISC- and proliferation genes28, and Prom1 (also known as CD133), a well-established colon cancer stem cell marker29.

PC cluster 4 consisted of cells from mice in which double (AK) and triple (AKP) mutations were targeted to PCs (23.25%11.23% and 52.26%28.23% of traced cells, respectively). Increased expression of Anxa2 (encoding annexin 2), a functional marker of inflammatory response, and Clu (clusterin), previously shown to earmark revival stem cells (RSCs) upon gamma-irradiation30, feature in PC cluster 4. Accordingly, evaluation of the RSC signature showed elevated expression among the PC clusters (Fig. 2e), and in situ hybridization analysis in PC-derived tumors from mice carrying compound mutations (AK and AKP) (Fig. 2f and Extended Data Fig. 4a,b) confirmed increased Clu expression. Finally, pathway analysis revealed the similarities between the PC-derived cluster 4 and RSCs, both earmarked by the activation of Yap1 signaling and specific inflammatory pathways (Fig. 2g). Compared with RSCs, PC-derived and Apc/Kras-mutant cells from cluster 4 showed increased levels of TGF and WNT signaling (Extended Data Fig. 4c).

Thus, upon genetic targeting or inflammatory stimulus, PCs escape their homeostatic identity and acquire distinct cellular features, as shown by scRNA-seq and FACS analysis (Extended Data Fig. 4d,e). Of note, DSS treatment led to lower expression of Lgr5 and Ascl2 in stem cells, as well as a lower association of the ISC signature, confirming our and others previous observations that resident stem cells lose their multipotency upon acute inflammation13 (Extended Data Fig. 4f).

Collectively, these findings demonstrate that PCs efficiently dedifferentiate upon genetic targeting or inflammatory stimulus leading to distinct cellular identities. During tumorigenesis driven by Apc/Kras, PCs share features with the YAP1-dependent RSC identity, and further activate TGF and WNT signaling in their conversion to bona fide tumor cells.

To investigate the consequences of cell-of-origin identity on the transcriptional profile of the resulting intestinal tumors, we performed bulk RNA sequencing (RNA-seq) of macroscopically dissected lesions originating from ISCs and PCs (Fig. 3a). Principal component analysis (PCA) revealed that the major variance component (61%) was attributed to differences in the cell-of-origin, whereas the impact of genotype or inflammatory stimulus became notable in the second component of variation (10%) (Fig. 3b). Differential expression analysis between tumors derived from PCs and ISCs in the same genetic and inflammatory context (Apc and DSS) revealed tumor signatures specific for each cell-of-origin (Supplementary Table 2).

a, Schematics of the experimental approach to compare PC- and Lgr5-derived adenomas. b, PCA plot showing that the cell-of-origin is the dominant discriminator of variance. Ctrl, control. c, Bar plot summarizing the GSEA between Paneth-derived (Lyz1 Apc DSS) and ISC-derived (Lgr5 Apc DSS) tumors. Pathways were filtered based on P<0.05 and absolute normalized enrichment score (NES)>0.5. Pathways that are significant in the IBD-CRC versus sCRC comparison have bold labels. ROS, reactive oxygen species. d, Subset of inflammatory pathways, visualized as a heatmap based on values from the GSVA. e, Box plots showing results of the stem cell index. The P value depicts the result of one-way ANOVA. n=3 biologically independent samples per group. Box plots display the median, lower and upper hinges corresponding to the first and third quartiles. Whiskers extend from the hinges to maximum or minimum values, no further than 1.5 interquartile range. f, GSEA showing a significant but opposite association between the Lyz1 tumor signature and IBD-CRCs, and between the Lgr5 tumor signature and sCRC. The P value denotes the BenjaminiHochberg adjusted value of the two-sided enrichment P value from GSEA. g, Heatmap showing GSVA scores, averaged per tumor group, of pathways with similar patterns between the mouse and human tumor groups. h, Heatmap highlighting differentially expressed genes (log2-transformed fold change>1.5, Padj<0.01. Two-tailed P values were derived from the Wald test with BenjaminiHochberg correction for multiple testing shared between the Paneth or IBD tumors and the Lgr5 or sCRC tumors. Values denote z-scores of average expression per cell type. cDC, conventional dendritic cells; EC, endothelial cells; NK, natural killer cells; Ig, immunoglobulin. im, Two distinct sporadic colon cancer identities become apparent upon analysis of a large cohort of CRC tumors (n=3,232 samples). i, Heatmap showing Pearson correlations of the GSVA scores. jk, Scatter plot showing two distinct clusters, sporadic-like and colitis-like, in all colon cancers (j) and stratified according to their CMS (k). Gray lines indicate contours lines, and dashed lines show thresholds to classify tumors in colitis-like, sporadic-like and intermediate groups. l, Stacked bar plot analysis showing the distribution of CMS1 to CMS4 across colitis-like and sporadic-like colon cancers. m, KaplanMeier survival analysis for relapse-free survival. P values denote the result of the log-rank test and Cox regression models for univariate analyses. Hazard ratios (HR) and confidence intervals (CI) are shown for pairwise comparisons.

Source data

Gene set enrichment analysis (GSEA) indicated that tumors derived from ISCs were characterized by high levels of MYC and WNT signaling, whereas PC-derived adenomas showed higher levels of inflammatory pathways indicative of infiltration from the tumor microenvironment (TME) (Fig. 3c and Extended Data Fig. 5). Of note, the inflammatory characteristics of PC-derived tumors were observed also in mice in which Apc and Kras mutations were targeted to PCs in the absence of DSS-driven inflammation (Fig. 3d), indicating that specific mutant genotypes and type of cell-of-origin can trigger tumor initiation by mimicking the inflammatory context otherwise brought about by DSS.

Next, we used the ISC index31 (Methods) to predict the relative proportions of RSC and crypt-base columnar (CBCs) stem cells in the intestinal tumors. In agreement with the scRNA-seq analysis, PC-derived tumors were RSC-enriched, whereas ISC-derived tumors consisted mainly of CBCs (Fig. 3e). Notably, the highest RSC contribution was observed in tumors originating from Apc/Kras-mutant PCs in the absence of inflammation, when compared with the equivalent genotype upon DSS administration. The latter is of relevance to dissect the relative contribution of inflammatory insult and somatic mutations in the dedifferentiation process leading to tumor initiation. As mentioned before, one of the primary effects of various forms of tissue injury to the intestinal epithelium is the loss of resident stem cells. In a study by Singh et al.32, ablation of Lgr5+ ISCs was performed to study its consequences using scRNA-seq. Our analysis of these datasets revealed elevated expression of secretory genes in regenerated stem cells normally restricted to the PC lineage (Extended Data Fig. 6a). In parallel, upon Lgr5+ cell ablation, PCs and goblet cells partially activate the RSC program (Extended Data Fig. 6b), indicating that stem cell loss is sufficient to trigger plasticity and RSC reprogramming from PCs. Hence, to assess whether removal of the resident stem cells is sufficient to activate the lineage-tracing capacity of PCs in the absence of inflammatory injury, we implemented a similar diphtheria toxin receptor (DTR)-based Lgr5+ ablation experiment33. As shown in Extended Data Fig. 6c,d, lineage tracing from PCs was similar to that previously shown upon inflammation13.

Together, these results indicate that the cell-of-origin embodies the major source of intertumor variability, and that the Paneth or ISC origin is reflected by the RSC- or CBC-like profile of the resulting tumors, respectively. Notably, inflammation-driven ISC loss activates dedifferentiation at the epithelial level.

The small intestinal location of PCs and the tumors originating from them raises questions on the relevance of our study for colon cancer, one of the most frequent causes of morbidity and mortality because of malignancy. To explore the general applicability of our results, we first analyzed bulk RNA-seq and scRNA-seq data from two distinct studies34,35 centered around the azoxymethane (AOM)/DSS mouse model of colitis-driven colon cancer36. This protocol relies on the oncogenic -catenin mutations caused by AOM, which in combination with DSS-driven ulcerative colitis, result in multiple adenocarcinomas in the distal colon. As shown in Extended Data Fig. 6e (CIBERSORTx37; Methods), analysis of the bulk RNA-seq data35 from AOM/DSS-derived colon tumors revealed an abundant subpopulation reminiscent of the RSC-like PC cluster 4. Moreover, analysis of scRNA-seq data35 confirmed that AOM/DSS-derived colon tumors, when compared with their sporadic counterparts, were distinct in terms of the qualitative and quantitative composition of their TME, namely a pronounced presence of infiltrating immune cells and tumor-associated fibroblasts (Extended Data Fig. 6f). Accordingly, tumor cells derived in the context of AOM/DSS share transcriptional similarity with Paneth-derived tumor cells and RSCs (Extended Data Fig. 6g,h).

Hence, notwithstanding the small intestinal location of the PC-derived tumors, their gene expression signatures and overall inflammatory TME profiles are characteristic of colitis-associated carcinoma in the mouse.

Next, in view of the marked differences in the transcriptional profiles between mouse intestinal tumors with distinct cells-of-origin, we questioned whether similar differences distinguish human sporadic colon cancers from those that arise in the context of IBD. RNA-seq profiles of human microsatellite stable (MSS) sporadic colorectal cancers (sCRC, n=38) and from patients with inflammatory bowel disease (IBD-CRC, n=14) were interrogated38. GSEA of the most differentially expressed genes (log2-transformed fold change>5, adjusted P (Padj)<0.01) from the mouse tumors (Lyz1 tumor signature, n=27 genes; Lgr5 tumor signature, n=40 genes) revealed a significant association between the Lyz tumor signature and IBD-CRC (NES=1.67, Padj=6.2103), whereas the Lgr5T profile was significantly associated with sCRC (NES=1.62, Padj=0.013) (Fig. 3f, Extended Data Fig. 7ac and Supplementary Table 3). Evaluation of the hallmarks from the molecular signature database39 revealed gene sets common to PC-derived tumors and IBD-related CRCs (interferon alpha/interferon gamma, inflammatory response, IL-6/IL-2 signaling, KRAS, complement, allograft rejection), and to Lgr5-derived tumors and sCRCs (MYC targets, G2M checkpoint, E2F targets and WNT -catenin signaling) (Fig. 3g and Extended Data Fig. 7d).

We then compared the differentially expressed genes from PC-derived versus Lgr5-derived mouse tumors and human IBD-CRC versus sCRC (Paneth/IBD-CRC, n=49 genes; Lgr5/sCRC, n=27 genes) and visualized their expression across different cell types based on a large scRNA-seq CRC study40 (Fig. 3h). Of note, the markers shared between PC-derived tumors and IBD-CRC were dominantly expressed in myofibroblasts (for example, ITGAM, SLC1A3), T cells (for example, SELPLG, LAX1) and stromal cells (for example, CHRDL1, RELN). By contrast, Lgr5-derived tumors/sCRC markers were mostly observed in epithelial cells (for example, HOXB6, HOXB8, HOXB9, AXIN2, ASCL2), indicating the difference in stromal composition among these tumors (Fig. 3h). Comparison of a set of gene signatures (Supplementary Table 4) in a large CRC cohort41 confirmed the presence of two distinct identities (Fig. 3i,j): a colitis-like identity enriched with RSCs and prevalent in consensus molecular subtype 4 (CMS4) (67%) and CMS1 (36%) tumors; and a sporadic-like identity enriched with CBCs and common in CMS2 (55%) and CMS3 tumors (52%) (Fig. 3k,l and Extended Data Fig. 7e). Survival analysis revealed significant differences in relapse-free survival between the sporadic- and colitis-like CRC groups (P=5.2105) (Fig. 3m). Thus, transcriptional signatures derived from small intestinal mouse tumors originating from PCs significantly overlap with those from human colon cancer that arose in the context of IBD, possibly revealing a common cell-of-origin in secretory lineages.

Next, we investigated whether the inflammatory profile observed in PC-derived tumors is earmarked by distinct immune cell populations. By means of gene set variation analysis (GSVA) with a tumor-infiltrating lymphocyte-specific gene signature42, a statistically significant higher tumor-infiltrating lymphocytes score was observed in PC-derived versus Lgr5-derived mouse tumors (Extended Data Fig. 8a). When using deconvolution of our data with CIBERSORTx37, a significantly higher score was observed for immune cells in PC-derived versus Lgr5-derived tumors (Extended Data Fig. 8b). Furthermore, when zooming in on defined immune cell types, a highly significant and unique enrichment of + T cells was observed in PC-derived tumors (Extended Data Fig. 8c,d), in contrast to the enrichment of regulatory T (Treg) cells in Lgr5-derived tumors. These findings were in concordance with what we observed for colitis-like versus sporadic-like CRCs (Extended Data Fig. 8e,f). These observations were further validated by analysis of the scRNA-seq data obtained from mouse colonic tumors induced by AOM/DSS previously35. As shown in Extended Data Fig. 9fh, a subpopulation of Cd8/Cd4 and Pdcd1+/ll17a+ T cells earmarks these tumors, which likely represent the counterpart of + T cells observed in colitis-like colon cancers.

The relative representation of patient-derived colon cancers whose expression profiles are reminiscent of the PC-derived mouse tumors (~25%; Fig. 3j) vastly exceeds the expected proportion of colon cancers arising in patients with an history of clinically manifest IBD (12%)43. One possible explanation for this apparent discrepancy may be that western-style dietary habits, often in combination with chronic over-nutrition and sedentary lifestyles, have been associated with a state of chronic metabolic inflammation, termed metaflammation44. In particular, a link between the consumption of a diet high in fat and sugar and PC dysfunction has recently been demonstrated45. Moreover, a purified mouse diet that mimics western-style dietary habits and underlies the increased risk of colon cancer (NWD1)46 was recently shown to induce a low degree of chronic intestinal inflammation and other mechanisms that define pathogenesis of human IBD47. Therefore, we hypothesized that etiological drivers of colon cancer other than IBD, including widespread western-style dietary habits, may underlie dedifferentiation and tumor onset mechanisms similar to those observed upon acute DSS-driven inflammation.

To provide support for this hypothesis, we first fed C57BL6/J mice for 3months with the western-style (NWD1) and control (American Institute of Nutrition 76A (AIN-76A)) diets and compared the transcriptional response of PCs with that obtained upon DSS administration (Fig. 4a). We examined genes upregulated upon inflammatory stimuli (DSS signature; Methods), which showed variable but overall increased levels in NWD1-fed mice when compared with those on the control diet (Fig. 4b). Indeed, GSEA confirmed that the DSS signature was associated significantly with PCs exposed to NWD1 (NES=2.99, Padj<0.001), indicating that western-style dietary habits trigger an inflammatory-like response in PCs (Extended Data Fig. 9a). At the gene ontology level, the western-style diet activated signaling pathways related to the cell cycle (G2M checkpoint) and proliferation (mitotic spindle, MYC targets; Extended Data Fig. 9b), suggesting that PCs re-enter the cell cycle upon long-term exposure to a western-style diet, similar to what is observed in DSS-driven inflammation13.

a, Schematics of the experimental approach designed to investigate the consequences of short- and long-term exposure to a western-style diet (NWD1) versus control (AIN-76A) diets. b, Heatmap showing z-scored DSS signature (DSS versus control; Padj<0.05, log2-transformed fold change>0.25) in PCs exposed to DSS or NWD1. c, Organoid multiplicities derived either from single ISCs or PCs, and from reconstituted doublets (L, Lgr5+ ISCs; P, PCs). Pooled data from n=4 independent experiments. P values were calculated using one-way ANOVA and Tukeys tests for group comparisons. Error bars depict s.d. d, Representative image of lineage tracings from a NWD1-fed Lyz1-YFP mouse. Scale bars, 50m. The P value depicts the result of a two-sided Students t-test and the error bars represent s.d. Data from n=3 mice. e, UMAP showing PCs from mice fed AIN-76A (AIN) and NWD1 (n=3 mice per condition). The DSS signature portrayed on UMAP embedding highlights a subcluster of PCs responsive to the NWD1 diet. f, Violin plots showing different levels of the DSS signature (top) and CytoTRACE score (bottom) between PCs responsive to the NWD1 diet and other PCs. P values show the significance of a two-sided Wilcoxon test. g, Violin plots representing marker genes of PCs responsive to the NWD1 diet, showing coexpression of stem and secretory markers. h, Heatmap visualization of GSVA, indicating pathways that are activated in PCs after exposure to DSS or NWD1. Comparison with data from ref. 47. i, UMAP plot of PC subset in the scATAC dataset of mice treated with AIN (n=2) and NWD1 (n=2). j, Heatmap listing differential peaks between diet response PCs and other PCs. k, Ideogram displaying the distribution along the mouse chromosomes (Chr) of the differential peaks observed upon diet response (red) when compared with those characteristic of PCs (blue).

Source data

In view of the previously reported acquisition of stem-like features by PCs upon inflammation13, we next used the organoid reconstitution assay (ORA)12,48 to assess whether similar effects are exerted by the western-style diet. We first coincubated Paneth and Lgr5+ cells from AIN-76A- and NWD1-fed mice in all four combinations. As shown in Fig. 4c, PCs from NWD1-fed mice significantly improved organoid formation independently of their reconstitution with Lgr5+ cells from NWD1- or AIN-76A-fed mice, possibly indicative of a paracrine effect enhancing the well-established niche (ISC-supporting) role of PCs. However, single (that is, nonreconstituted with Lgr5+ ISCs) PCs from NWD1-fed mice formed organoids more efficiently when compared with either PCs from AIN-76A-fed mice or with Lgr5+ ISCs from both groups of mice. These ex vivo results were further validated by lineage-tracing analysis of PCs in R26LSL-YFPLyz1CreERT2 NWD1-fed mice that revealed extended yellow fluorescent protein (YFP)-labeled ribbons thus confirming their dedifferentiation and acquisition of stem-like features induced by the western-style dietary cues (Fig. 4d).

To focus in on the primary transcriptional response of PCs to NWD1, we took advantage of the scRNA-seq data generated previously47 upon exposure to NWD1 (Fig. 4a). Within 4days of switching mice to the NWD1 diet, a subset of PCs became apparent whose transcriptional profile was strongly associated with the DSS signature (labeled diet response cells in Fig. 4e). Mirroring our previous observations obtained immediately upon DSS inflammatory stimulus, these WSD-responsive cells increased their transcriptomic diversity as measured by CytoTRACE49 (Fig. 4f and Methods). After short exposure to NWD1, the diet-responsive cells acquired stem cell markers while retaining some secretory features (Fig. 4g). Comparative pathway analysis between the transcriptional response of PCs to DSS and NWD1 revealed similar upregulation of the WNT, MYC, Hedgehog and G2M checkpoint signaling pathways (Fig. 4h).

Because the observed western-style diet-driven changes in gene expression are likely exerted through epigenetic modifications, we next analyzed scATAC (single cell assay for transposase-accessible chromatin with high-throughput sequencing) data obtained in the framework described previously47. Similar to what was observed by scRNA-seq analysis, we identified a group of NWD1 diet-responsive PCs with significant epigenetic modifications, including a main cluster (52%) on mouse chromosome 11 (synthenic with human chromosomes 17 and 5) that encompasses a considerable fraction of the genes encoding for members of the WNT, PI3KAKT and cell-cycle pathways (Fig. 4ik and Extended Data Fig. 9cf). The latter were previously shown by our laboratory to underlie PC dedifferentiation and the acquisition of stem-like features upon DSS-driven inflammation13.

To assess the effects of the NWD1 diet on mouse colon, we searched for proliferating Paneth-like cells, also known as DCS cells9,12. Inflammation-driven cell-cycle activation in these allegedly postmitotic lineages was previously observed in small intestinal PCs of DSS-treated mice and of patients with Crohns disease13. By co-staining colonic tissues with the secretory lineage marker wheatgerm agglutinin (WGA)50 and Ki67, a significant increase in the number of proliferating secretory cells located at the crypt bottom was observed both in mice fed the NWD1 diet and, as a positive control, in those administered DSS in their drinking water (Fig. 5ac).

a, Colonic tissues from either untreated mice or mice administered 3% DSS for 7days, as well as from mice fed AIN-76A or NWD1 synthetic diets for 3months were analyzed for the presence of proliferative DCS cells. WGA was used to stain DCS cells50, and Ki67 to mark proliferative cells. Tissues were counterstained by DAPI (nucleus). Asterisks mark WGA/Ki67 double-positive cells. b,c, Quantification of WGA+Ki67+cells (b) and total WGA+ cells (c) in the lower colonic crypt of the mice, as shown in a. Scale bar, 20m. A minimum of 50 crypts from three different mice were analyzed. Data are presented as mean and s.d. P values denote two-tailed Tukeys tests for group comparisons. d, Bar plot showing the predicted cell-of-origin in IBD-CRC (n=25) and sCRC (n=257)38 cohorts based on the COOBoostR computational approach53 (Methods). The P value is the result of Fishers exact test. e, MUC2, BEST4 and Ki67 IHC analysis of colonic tissues obtained from controls and patients with IBD. Asterisks indicate double-positive cells. Scale bars, 50m. f, Box plot showing percentage of cycling (Ki67+) MUC2+ and REG4+ cells in patients with ulcerative colitis (UC) and controls. scRNA-seq data are from ref. 55. Positivity was defined per cell by the presence of at least one read for that particular marker. Subsequently, cells were aggregated per patient to calculate percentages. n=12 healthy participants and n=17 patients with ulcerative colitis. Box plots show the median, and lower and upper hinges correspond to the first and third quartiles. Whiskers extend from the hinges to maximum and minimum values, no further than 1.5 interquartile range. The P value shows the result of a two-sided t-test. g, Box plot denoting differences in the stem cell index based on stratification of predicted cell-of-origin in a subset of IBD-CRC and sCRC cases for which RNA-seq data were available (n=27 stem, n=10 goblet)38. The P value shows the result of a two-sided t-test. Box plots display the median, and lower and upper hinges corresponding to the first and third quartiles. Whiskers extend from the hinges to maximum and minimum values, no further than 1.5 interquartile range. h, Mapping of CMS on tumor samples stratified according to their predicted cell-of-origin. The P value shows the result of Fishers exact test. i, Graphic abstract of the model arising from this study. Colon cancer can be initiated either from stem (ISC) or differentiated cells, the latter in response to inflammatory cues. RSC reprogramming is activated in support of the regenerative response. During this process, actively dividing RSCs expand the cell targets for tumor initiation and progression, leading to an alternative route to tumorigenesis earmarked by an inflammatory phenotype.

Source data

Collectively, our results reveal an alternative bottom-up route to intestinal tumorigenesis originating from PCs in the mouse small intestine and, allegedly, from secretory lineages in the human colon, triggered by inflammatory cues, as in IBD, or through western-style dietary factors. To validate the relevance of our mouse study in patient-derived colon cancer, we took advantage of new computational methods51,52,53 developed to predict the cell-of-origin of tumors by matching the mutational density along the cancer genome with the profiles of epigenetic modifications characteristic of normal cell types54 (Extended Data Fig. 10a). Although tumors from patients with a history of IBD have a similar genome-wide tumor mutational burden compared with their sporadic counterparts (Extended Data Fig. 10b), the presence of regions (genomic windows, Methods) that are differentially mutated between sCRC and IBD-CRC is suggestive of alternative mutational patterns (Extended Data Fig. 10c,d). Hence, we compared the individual mutational landscapes with the epithelial cell types of the colon to compute the putative cells-of-origin.

As shown in the Fig. 5a (COOBoostR53, Methods), although the majority of the sporadic colon cancers appear to originate from stem cells (52%), among the IBD-related cases, goblet (40%) and BEST4 (28%) cells represent the prevalent cells-of-origin. Strikingly, a substantial proportion (>40%) of sporadic cases are also predicted to originate from non-stem lineages, namely goblet (22%), enterocytes (2%) and BEST4 cells (17%). The latter, named after the specific expression of the bestrophin 4 gene (BEST4), form a newly identified and as yet only partially characterized intestinal epithelial lineage with dual absorptive and secretory features55,56,57. IHC and scRNA-seq analysis in a cohort of patients with ulcerative colitis confirmed the presence of actively proliferating goblet and BEST4 cells (MUC2+ and BEST4+) (Fig. 5e,f), indicative of the primary effects of inflammation in these otherwise postmitotic cells, likely to precede their acquisition of stem-like features.

These results are indicative of the fraction of sporadic colon cancers whose expression profiles are reminiscent of the PC-derived tumors and IBD-CRCs (24.7%; Fig. 3j). Furthermore, the availability of RNA-seq data from a subset of the patient-derived tumors allowed us to confirm the prevalence of RSC-like expression profiles and the high proportion of CMS1 and CMS4 among the sporadic and IBD cases predicted to originate from non-stem, and in particular goblet, cells (Fig. 5g,h and Extended Data Fig. 10e).

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Single-cell RNA-sequencing of hematopoietic progenitor cells of del(5q) MDS patients

To identify the transcriptional alterations characterizing hematopoietic progenitors harboring del(5q), we initially performed scRNA-seq of CD34+ cells of four newly diagnosed patients with del(5q) MDS (Patient_1-4), and three age-matched healthy donors (Healthy_1-3) using the 10X Genomics technology (Fig.1A) (gating strategy can be found in Supplementary Fig.1). The clinical and genomic characteristics of the MDS patients and healthy donors are shown in Supplementary Table1. The percentage of cells with del(5q) based on the cytogenetic analysis varied between 35 to 90%. In all cases, the common deleted region encompassed bands 5q(13-33) (genes shown in Supplementary Data1).

A CD34+ cells were obtained from bone marrow aspirates of newly diagnosed del(5q) MDS patients, healthy donors and patients treated with Lenalidomide, and were subjected to single-cell RNA sequencing and analysis. PCR partial cytogenetic responder, CCR complete cytogenetic responder, NR non-responder. Part of this figure was created with BioRender.com. B Uniform Manifold Approximation and Projection (UMAP) of 42,494 cells representing the expected 14 hematopoietic progenitors: HSC hematopoietic stem cells, LMPP lymphoid-primed multipotent progenitors, GMP granulocyte-monocyte progenitors; granulocyte progenitors; monocyte progenitors; dendritic cell progenitors; CLP common lymphoid progenitors; B-cell progenitors; T-cell progenitors; MEP megakaryocyte-erythroid progenitors; MK_Prog megakaryocyte progenitors; early erythroid progenitors; late erythroid progenitors; basophil progenitors. C Per patient UMAP showing the identity of the cells projected from the integrated space. D Dotplot showing the percentage and value of the normalized expression of the canonical marker genes used to assign the cell identity to each cluster. E Barplot representing the contribution of cells from each patient to the different cell types. F Barplot representing the number of cells assigned to each cell type for the studied patients. G Barplot representing the percentage of cells assigned to each cell type for del(5q) MDS patients and healthy samples. N=7 biologically independent samples were used (n=3 healthy donors and 4 del(5q) MDS patients). Data are presented as mean values +/SD. Two-sided Wilcoxon signed-rank test was used to calculate statistical significance. Exact p-values for the differential abundance of each hematopoietic progenitor between the del(5q) MDS and the healthy condition were the following: HSC: p=0.63; LMPP: p=0.23; GMP: p=0.06; Granulocyte: p=0.23; Monocytes: p=0.06; DendriticCell: p=0.63; CLP: p=1; pro-B: p=0.23; T: p=0.04; MEP: p=0.11; MK_Prog: p=0.63; EarlyErythroid: p=1; LateErythroid: p=0.23; Basophil: p=0.4.

A total of 55,119 and 45,311 cells from patients and healthy donors, respectively, were profiled and integrated. After applying quality filters, 46,772 and 43,442 cells were eventually included in the downstream analysis. Data was integrated, clustered and manually annotated (Fig.1B, C, Supplementary Fig.2A, B) based on curated markers (Fig.1D, Supplementary Fig.2C), obtaining 14 and 13 clusters (patients and donors, respectively) representing all the expected hematopoietic progenitor subtypes. Contribution of every MDS patient and donor to the composition of all the clusters was identified (Fig.1E, Supplementary Fig.2D), and each individual showed different proportions of hematopoietic progenitors (Fig.1F, Supplementary Fig.2E). Although there were some differences in the percentage of hematopoietic progenitors between MDS patients and healthy donors (e.g., HSC), these differences were not statistically significant which might be related to the high variability in cell composition across samples (Fig.1G).

Identifying single-arm copy number variations (CNVs) at the single-cell level presents challenges due to potential compensatory mechanisms of alleles, as well as to the sparse and noisy nature of single-cell data. In this study, we employed two different and complementary approaches: CopyKat23 (Fig.2A), which relies on gene expression, and CaSpER24 (Fig.2B), which relies on allele frequencies (see Methods). This combined strategy aimed to enhance the sensitivity and accuracy of identifying cells harboring 5q deletion. To avoid false positive detection, we only classified the cells as harboring the del(5q) if the same cell was characterized as such by the two different algorithms (Fig.2C). To validate this classification, we analyzed the expression pattern of genes encoded in the deleted region in individual cells. Due to the sparsity of scRNA-seq data, we were only able to detect six genes as highly variable, CD74, RPS14, BTF3, COX7C, HINT1 and RPS23, whose expression was decreased in del(5q) when compared to non-del(5q) cells at sample level (Fig.2D), further confirming our del(5q) cell classification. Once the classification was performed, we applied a Wilcoxon signed-rank test between cells classified as del(5q) and non-del(5q), revealing in the underexpressed fraction of the genes an enrichment for the genes located on the deleted locus (Supplementary Fig.3A, B). To further validate the classification, we randomly shuffled the labels from the classified cells, and repeated the same differential expression analysis, revealing how the genes located on the deleted region started to fade away (Supplementary Fig.3C). Based on this classification, interestingly, for each individual patient, the proportion of del(5q) in the CD34+ progenitor cells was consistent with that obtained by karyotype in total bone marrow (Fig.2E).

A Heatmap of the results of CopyKat showing the copy number alteration score given to each 200kb bins in chromosome 5. In order to represent cells, a clustering has been performed within each sample (kmeans with k=80), and a posterior clustering has been applied to detect the clusters containing the cells harboring the deletion. The control sample used by the algorithm is an MDS sample with normal karyotype, while the healthy sample with normal karyotype represents an additional negative control for the analysis. B Barplot representing the percentage of cells inferred by CaSpER that harbor an amplification, a deletion or a normal number of copy number variation in each branch of chromosome 5 per patient. The control corresponds to an MDS sample with normal karyotype, which is used as a reference by the algorithm. C Venn diagram representing the number and percentage of cells classified as del(5q) by both algorithms. D Pseudobulk normalized expression of the 6 CDR-genes with higher expression in our dataset (CD74, RPS14, BTF3, COX7C, HINT1 and RPS23) separated by genotype. N=4 biologically independent samples were used. The number of del(5q) and non-del(5q) cells were used to generate the pseudobulks for each patient can be found in the Source Data. E Graph depicting the percentages of del(5q) cells inferred by karyotype, CaSpER and CopyKat for each patient. Selected cells correspond to the cells classified as del(5q) cells by both computational algorithms.

We then interrogated the distribution of del(5q) cells across the different hematopoietic progenitors. Cells with the deletion were detected in all the defined hematopoietic progenitor clusters (Fig.3A), although a high heterogeneity of distribution was observed among patients (Fig.3B, C). Despite the observed heterogeneity, a statistically significant accumulation of del(5q) cells was detected in early erythroid progenitors across all individuals (hypergeometric test, p-value<0.05). Additionally, three out of four patients exhibited statistically significant enrichment of del(5q) in granulocyte-monocyte progenitors (GMP), megakaryocyte and late erythroid progenitors (Fig.3D). Collectively, these results indicated a bias of del(5q) cells towards specific myeloid compartments, mainly towards erythroid cells, which is consistent with the association between this genetic lesion and the anemia that characterizes patients with del(5q) MDS.

A UMAP representing all the MDS samples integrated and colored by cell type. The density map represents the distribution of the cells classified as del(5q) by the two algorithms. B UMAPs with density maps representing the distribution of del(5q) cells per individual patient. C Barplots showing the number of del(5q) and non-del(5q) cells composing each cell type for each MDS patient. D Heatmap representing the enrichment of del(5q) cells (-log10(p-value)) in each cell type. Any color different from white represents a statistically significant enrichment of del(5q) cells (p-value<0.05). p-values were calculated using the one-sided hypergeometric test. The number of biologically independent replicates (cells) used for the hypergeometric test and the exact enrichment p-values can be found in the Source Data.

To delve into the transcriptional program associated with del(5q) cells in patients with MDS, we performed a pseudobulk differential expression (DE) analysis between del(5q) and non-del(5q) cells for each cell population, as traditional Wilcoxon signed-rank test -based DE analysis in single cell data has recently shown to yield high false positive rates25. Intriguingly, considering every type of hematopoietic progenitors, only seven genes were differentially expressed (downregulated) in del(5q) in comparison with non-del(5q) cells (Fig.4A). Some of the downregulated genes played a key role in MDS and other non-hematological tumors, such as PRSS21, which encodes for a tumor suppressor frequently hypermethylated in cancer26, MAP3K7CL, whose downregulation serves as a biomarker in other types of cancer27, and CCL5, whose downregulation is associated with high-risk MDS28.

A Heatmap representing the differentially expressed genes (BenjaminiHochberg-adjusted p-values<0.05 and |logFC|>2) between del(5q) and non-del(5q) cells within each hematopoietic progenitor. The heatmap was created by combining n=4 del(5q) MDS patients and generating pseudobulks per cell type. The two-sided edgeRs Likelihood Ratio Test was used to calculate p-values. The exact number of biologically independent replicates (del(5q) and non-del(5q) progenitor cells), as well as the specific p-values for each differentially expressed gene can be found in the Source Data. B Dotplot representing statistically significant biological processes and pathways (BenjaminiHochberg-adjusted p-values<0.05) for differentially expressed genes obtained in del(5q) versus Healthy and the non-del(5q) versus Healthy contrasts. The one-sided hypergeometric test was used to calculate p-values. Del(5q) and non-del(5q) cells were derived from n=4 del(5q) MDS patients, whereas healthy cells were derived from n=3 healthy donors. Biologically independent replicates (del(5q), non-del(5q) and healthy progenitor cells) used for each comparison are specified in the Source Data. CE Histograms representing the activity score in all the cells separated by conditions. Some regulons behaved similarly in the MDS samples (non-del(5q) and del(5q) cells) compared to healthy cells (C), while other regulons behaved differently in the three different conditions (D). Some inferred regulons had an activity score on the MDS samples, while lacking on the healthy samples (E).

Due to the unexpected transcriptional similarity between del(5q) and non-del(5q) cells within MDS patients, we next performed a DE analysis between del(5q) MDS cells and CD34+ cells from healthy donors. This comparison yielded 20 to 988 differentially expressed genes (FDR<0.05 and |logFC|>2), depending on the progenitor cell (Supplementary Fig.4A, Supplementary Data2). Although most of these genes were cell-type-exclusive, they were enriched in similar pathways in most of the cell types (Fig.4B, left panel). Genes overexpressed in del(5q) cells were enriched in cell cycle and mitosis-related signatures, such as DNA replication and mitotic nuclear division, and showed increased expression of DNA repair related genes, suggesting that loss of 5q confers increased proliferative potential. Additionally, del(5q) erythroid progenitors, LMPPs, GMPs, DCs, and monocyte progenitors showed significant upregulation of genes involved in the p53 signaling and, genes involved in the apoptosis pathway were significantly upregulated in LMPP, MEP and late erythroid progenitors, but not in early erythroid progenitor cells. Our results are in line with the increased levels of apoptosis described for del(5q) patients29,30,31. Downregulated genes showed enrichment in ribosomes and translation related pathways in all hematopoietic progenitors, in line with previous works that have described del(5q) MDS as a ribosomopathy2,30,32. Interestingly, besides the cytoplasmic translation, we also observed altered expression of genes associated with mitochondrial translation altered in HSCs, GMPs and granulocyte progenitors. The comparison of non-del(5q) and healthy cells resulted in 64-736 altered genes per progenitor (Supplementary Fig.4B, Supplementary Data2). Enriched processes were also homogeneous among most hematopoietic progenitors and, as expected, were similar to the ones observed in del(5q) vs healthy comparison (Fig.4B, right panel).

Despite the low number of DE genes between del(5q) and non-del(5q) cells, we were interested in understanding whether differences in GRN might be observed between these two populations. Unlike DE analysis, which is performed in a gene-by-gene manner, GRN studies use data-driven grouping of genes to enable the identification of mechanistic transcriptional differences between conditions. Thus, we applied SimiC33 to compute the regulatory activity of regulons and observed that although some regulons behaved uniformly (low regulatory dissimilarity score, in Supplementary Fig.5A black-purple color) between the three conditions (del(5q), non-del(5q) and healthy cells), a group of regulons showed differential activity (high regulatory dissimilarity score, in Supplementary Fig.5A yellow-orange color) across the conditions. Among them, three different regulon activity patterns arose. Firstly, a group of regulons that showed similar activity between non-del(5q) and del(5q) cells, and different to healthy cells, in line with DE analyses, such as the ones driven by ZNF451, YBX1 and PSPC1 (Fig.4C). Secondly, there were regulons with differential activity between the three conditions, such as those driven by JARID2, IRF1 and KAT6B, among others (Fig.4D). The three regulons showed high activity in cells from healthy age-matched controls (6184 years), whereas they presented a progressively lower activity in non-del(5q) cells, and their lowest activity in del(5q) cells. JARID2 acts as a tumor suppressor and plays a crucial role in the leukemic transformation of myeloid neoplasms34, and its deletion promotes an ineffective hematopoietic differentiation35, suggesting that the low activity of this regulon may negatively impact the hematopoietic differentiation of these patients. IRF1 is located in 5q31.1 and its deletion in one or both alleles has been observed in MDS and AML patients with chromosome 5 abnormalities36. IRF1 has been described as a master HSC regulator, and its loss impairs HSC self-renewal and increases stress-induced cell cycle activation, suggesting that its low activity in patients could confer proliferative advantage37. Decreased expression of KAT6B in aged hematopoietic stem cells has been associated with impaired myeloid differentiation38, suggesting that its almost non-existent activity in del(5q) cells may contribute to aberrant differentiation of these cells. Lastly, we detected regulons exhibiting differential activity between del(5q) and non-del(5q) cells and that showed no activity in healthy cells. In particular, regulons driven by RERE and KDM2A showed higher activity in del(5q) cells than in non-del(5q) cells (Fig.4E). RERE negatively regulates the expression of target genes, and such genes are enriched in cytoplasmic translation, ribosome biogenesis and ribonucleoprotein complex biogenesis pathways, among others (Supplementary Fig.5B). The KDM2A regulon was enriched in protein stabilization, regulation of cellular protein catabolic process and regulation of protein stability (Supplementary Fig.5B). The association of KDM2A and ribosomal genes has been already described by previous studies, postulating that KDM2A overexpression reduces the transcription of rRNA39,40.

Altogether, our results suggest a low transcriptional impact of 5q loss, with del(5q) and non-del(5q) cells presenting very similar gene expression alterations when compared to healthy controls, with such alterations being involved in processes that could contribute to abnormal hematopoietic differentiation. Nevertheless, although limited in number, genes and regulons specifically altered in del(5q) cells, such as those driven by JARID2, KAT6B, RERE or KDM2A, seem to be relevant for proliferation and myeloid differentiation, supporting the concept that cells harboring the deletion may have a more prominent role in the promotion of altered hematopoiesis.

To investigate whether the 5q deletion has a detrimental effect on cell-cell interactions between CD34+ progenitors, thus contributing to disease development, we performed a cell-to-cell communication analysis using Liana41 in both del(5q) and healthy controls datasets. We identified 4,534 interactions in healthy controls, and 314 interactions that were common to all del(5q) MDS patients, most of them overlapping with those found in healthy cells (Fig.5A). Despite this strong overlap, several differences between del(5q) MDS and healthy individuals were detected: in patients, monocyte progenitors were the most communicative cells, interacting mainly with early erythroid progenitors (Fig.5B). However, in healthy donors, HSCs, GMPs, DC, monocyte and granulocyte progenitors were the most interactive compartments, with a notable communicative pattern between granulocyte and GMP/DC progenitors (Fig.5C). Furthermore, genes involved in these differential interactions were overrepresented in different biological processes in each phenotype. For instance, interactions driven by healthy hematopoietic progenitors were enriched in negative regulation of apoptosis, HSC proliferation, leukocyte/DC differentiation, and hemopoiesis, whereas those found in MDS progenitors were enriched in negative regulation of translation, oncogenic MAPK signaling and HIF-1 signaling (Fig.5D). Focusing on interactions driven by del(5q) and non-del(5q) cells within the patients (Fig.5B), we observed very subtle differences regarding the communicational pattern and the number of interactions observed for each of the compartments, and there were no interactions specifically established between del(5q) cells, corroborating the high similarity already described between del(5q) and non-del(5q) cells. Overall, our results are consistent with the previously described lack of significant differences in gene expression between del(5q) and non-del(5q) cells, suggesting that deregulation of hematopoiesis in patients with 5q MDS affects all CD34+ cells.

A Venn diagram showing the number of unique interactions in del(5q) MDS and healthy samples. Healthy unique interactions were considered as those present in at least one of the healthy individuals, while MDS unique interactions were those that were present in all the patients. Interactions were inferred from n=4 del(5q) MDS patients and n=3 healthy donors. B Heatmap depicting the number of interactions triggered by del(5q) and non-del(5q) MDS cells, C as well as those established among healthy hematopoietic progenitors. The Source represents the cell types that express the ligand, whereas the Target represents the cells that express the receptor. D Dotplot representing statistically significant biological processes and pathways (BenjaminiHochberg-adjusted p-value<0.05) in which are enriched the encoding genes taking part in the healthy and MDS interactions. The one-sided hypergeometric test was used to calculate p-values, whose exact values can be found in the Source Data. E Chord diagram representing the unique MDS interaction AGTRAP-RACK1 among different del(5q) and non-del(5q) progenitors. F Chord diagram depicting the unique healthy interaction HMGB1-CXCR4 established by healthy hematopoietic progenitors.

To uncover specific interactions that may contribute to the disease, we next focused on those interactions that had been gained or lost in MDS versus controls. There were 17 interactions identified in patients that were totally absent in healthy individuals, suggesting that additional communications arise when developing the disease. For instance, AGTRAP expressed in monocyte and late erythroid progenitors interacted with RACK1 in HSCs, LMPPs, MEPs, pro-B and basophil progenitors (Fig.5E). AGTRAP is known to be implicated in hematopoietic cell proliferation and survival42, whereas RACK1 has been postulated as a potential therapeutic target for promoting proliferation in other myeloid neoplasms43,44. The fact that these molecules are highly expressed in MDS could potentially be contributing to the enhanced proliferation observed in MDS cells. In contrast, there were 37 interactions that appeared in the healthy donors and were absent in the patients, including the one established between HMGB1 expressed in CLPs, DC, granulocyte, basophil, megakaryocyte, early erythroid and late erythroid progenitors, and CXCR4 present in HSCs (Fig.5F). HMGB1-CXCR4 interaction is known to trigger the recruitment and activation of inflammatory cells in tissue regeneration45,46, thus its loss could have a negative impact on the bone marrow niche. In summary, these analyses may allow the identification of potential interactions implicated in the pathogenesis of the disease that could represent new therapeutic targets.

We next aimed to understand the effect of treatment with the standard-of-care, lenalidomide, on the transcriptional alterations observed in del(5q) and non-del(5q) cells. We performed scRNA-seq on CD34+ cells of two patients (Patient_5-6), which had achieved hematological response (one with partial cytogenetic response (PCR), and the other one with complete cytogenetic response (CCR), respectively) (clinical information in Supplementary Table1). Data were integrated, clustered, manually annotated, and del(5q) cells were identified as described before (Fig.6A, B). Patients showed different percentages of del(5q) cells which were consistent with karyotype results (Fig.6C): the patient with PCR showed 1939 cells with del(5q) (37.13%), whereas the patient showing CCR presented only 11 cells with del(5q) after treatment (0.15%), validating the persistence of del(5q) progenitor cells at the time of complete clinical and cytogenetic remission9. Similar to what we observed at diagnosis, the distribution of del(5q) cells was heterogeneous among patients (Fig.6A, B), and both responders exhibited a statistically significant del(5q) enrichment in GMPs and erythroid progenitors. Interestingly, patient with PCR also showed an enrichment in LMPPs, megakaryocyte, monocyte, and granulocyte progenitors (Fig.6D).

A UMAP depicting the del(5q) density across the different hematopoietic progenitors obtained in three patients after lenalidomide treatment. HSC hematopoietic stem cells, LMPP lymphoid-primed multipotent progenitors, GMP granulocyte-monocyte progenitors; granulocyte progenitors; monocyte progenitors; dendritic cell progenitors, CLP common lymphoid progenitors; B-cell progenitors; T-cell progenitors, MEP megakaryocyte-erythroid progenitors, MK_Prog megakaryocyte progenitors; early erythroid progenitors; late erythroid progenitors; basophil progenitors. B Barplots showing the number of del(5q) and non-del(5q) cells composing each cell type for each MDS patient. C Percentage of the cells identified as del(5q) by karyotype, CASPER, CopyKat, and the selection by intersecting the two algorithms. D Heatmap representing the enrichment of del(5q) cells (log10(p-value)) in each cell type. Any color different from white represents a statistically significant enrichment of del(5q) cells (p-value<0.05). P-values were calculated using the one-sided hypergeometric test. The number of biologically independent replicates (cells) used for the hypergeometric test and the exact enrichment p-values can be found in the Source Data.

We have demonstrated in the previous analyses that at diagnosis both del(5q) and non-del(5q) progenitors displayed transcriptional profiles linked to an aberrant hematopoiesis. Since both PCR and CCR patients were in hematological response, we hypothesized that the remaining CD34+ cells after lenalidomide treatment, which are mainly composed of non-del(5q) progenitors, must be able to promote improved hematopoiesis and thus restore the transcriptional profile of normal progenitor cells. To demonstrate that lenalidomide, besides the potential apoptosis of del(5q) cells, could reverse transcriptional alterations harbored by non-del(5q) cells in responder patients, we performed a DE analysis between non-del(5q) cells from the CCR and PCR and the four patients at diagnosis. This comparison revealed significant transcriptional changes after lenalidomide treatment, resulting in 6223609 genes in the different progenitor populations for the CCR (Supplementary Fig.4C, Supplementary Data2) and between 4582409 genes for the PCR (FDR<0.05 and |logFC|>2) (Supplementary Fig.4D, Supplementary Data2). Note that these transcriptional differences are significantly greater than those related to patient heterogeneity at diagnosis (see previous sections), indicating that most uncovered altered genes after treatment are probably due to treatment effect rather than patient heterogeneity. Genes altered upon treatment were enriched in ubiquitination and proteasome-mediated catabolic processes, and in phosphatidylinositol related pathways, which is in line with the mechanism of action described for lenalidomide in MDS patients47,48. Moreover, we detected an enrichment in autophagy-related processes. Overall, our results suggested an increase of the two most important protein degradation pathways in non-del(5q) cells upon lenalidomide treatment (Fig.7A, first and second panels, Supplementary Table2). Furthermore, hematopoietic progenitors exhibited an increased expression of genes involved in erythroid differentiation and erythropoietin signaling after treatment (Fig.7B), validating the enhanced erythropoiesis in response to treatment19. Our analyses also detected a positive enrichment of PD-L1 expression and PD-1 checkpoint in non-del(5q) cells of the responder patients after treatment, suggesting a potential immunosuppressive mechanism of these cells in response to lenalidomide (Fig.7A, first and second panels, Supplementary Table2).

A Dotplot representing statistically significant biological processes and pathways (BenjaminiHochberg-adjusted p-value<0.05 and |logFC|>2) for differentially expressed genes obtained in different comparisons: non-del(5q) cells of the complete responder vs at diagnosis (1st panel); non-del(5q) cells of the partial responder vs at diagnosis (2nd panel); del(5q) cells of the partial responder vs the non-responder (3rd panel); non-del(5q) cells of the complete responder vs healthy cells (4th panel); non-del(5q) cells of the partial responder vs healthy cells (5th panel). For p-value calculation, one-sided hypergeometric test was used. Specific p-values for statistically significant biological processes can be found in the Source Data. The detailed breakdown of the grouped processes shown can be found in Supplementary Table2. B Boxplot showing the normalized expression of erythroid differentiation-related genes for non-del(5q) cells in MDS at diagnosis (n=4) or after treatment with lenalidomide (partial responder, n=1; complete responder, n=1). Biologically independent replicates (cells) for hematopoietic progenitors were: Early Erythroid: n=5196 (MDS at diagnosis); n=2200 (Partial Responder); n=3496 (Complete Responder); Late Erythroid: n=7168 (MDS at diagnosis); n=1056 (Partial Responder); n=1880 (Complete Responder); MEP: n=3108 (MDS at diagnosis); n=824 (Partial Responder); n=844 (Complete Responder). Two-sided Wilcoxon signed-rank test was used to calculate p-values, that were then BenjaminiHochberg-adjusted. Box plots indicate median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points). Exact p-values are shown within the figure. C Graphs representing the activity scores of proliferation and differentiation-associated transcription factors in healthy cells and non-del(5q) cells of MDS patients at diagnosis and after lenalidomide treatment, as well as D in del(5q) cells of MDS patients at diagnosis, with a partial response and with no response to lenalidomide. Specific activity scores can be found in the Source Data.

GRN analyses evidenced that some of the alterations described at diagnosis were potentially reverted after treatment in non-del(5q) cells. IRF1, the master HSC regulator located in 5q31.1, which showed abnormally low activity at diagnosis in non-del(5q) cells, showed an increased activity after treatment in both patients, with the PCR not reaching the activity level seen for healthy cells, and the CCR showing an augmented activity comparable to the healthy cells (Fig.7C). KAT6B, whose lower expression has been associated with impaired myeloid differentiation, showed an augmented activity in both patients despite not reaching the activity level of healthy cells. Finally, CUX149, a TF frequently mutated in myeloid malignancies and whose knockdown leads to an MDS-like phenotype, presented similar activity in non-del(5q) cells, showing higher activity than at diagnosis (Fig.7C).

Importantly, although some transcriptional lesions were reverted upon lenalidomide treatment, non-del(5q) cells continue exhibiting altered expression of ribosome-related genes, showing a negative enrichment of processes related to ribosomes, translation, and mitochondrial translation when compared to healthy cells (Fig.7A, fourth and fifth panels, Supplementary Fig.4E, F, Supplementary Data2 and Supplementary Table2). After treatment, early and late erythroid non-del(5q) progenitors from responding patients showed no statistically significant changes in these pathways. Moreover, GRN analyses detected groups of regulons with similar activity for non-del(5q) cells at diagnosis and after treatment response, but with a different activity to the healthy cells, indicating that lenalidomide did not affect their aberrant activity. Some examples included the tumor suppressor JARID234,35, ZNF451, a TF whose high expression in leukemic cells has been associated with poor outcome50, and NCOR1, a regulator of erythroid differentiation51 (Fig.7C). Moreover, non-del(5q) cells exhibited, both at diagnosis and after treatment, abnormal high activity of two regulons that were not active in healthy cells: ADNP and SMARCE1 (Fig.7C). Globally, these results indicate that treatment with lenalidomide has the potential to revert some of the transcriptional alterations present at diagnosis in non-del(5q) cells at least in patients that responded to lenalidomide. Nevertheless, some of the transcriptional alterations present at diagnosis were not modified which could be relevant for abnormal hematopoiesis, and potentially, for the future relapse of the patients.

In line with what has been observed in non-del(5q) cells from the PCR, the remaining del(5q) cells generally exhibited significant upregulation of genes involved in ubiquitin and phosphatidylinositol signaling and, autophagy and apoptosis pathways when compared to del(5q) cells at diagnosis (Supplementary Figs.4G,6A, Supplementary Data2 and Table3), which is consistent with the mechanism of action of lenalidomide47,48. However, these cells showed reduced expression of genes implicated in ribosomal and mitochondrial translation compared to diagnosis, along with diminished expression of DNA repair associated genes. (Supplementary Fig.6A). This suggests that lenalidomidedoes not fully reverse key transcriptional alterations that may underlie the ribosomopathy characterizing the disease.

Finally, to understand the transcriptional alterations associated with a lack of hematological response after lenalidomide treatment, we performed scRNAseq on CD34+ cells of an additional patient (Patient_7), who was refractory to lenalidomide (non-responder, NR). Data were processed as described previously (clinical information in Supplementary Table1), showing 83.8% of del(5q) cells (Fig.6AC), with a statistically significant increased abundance in LMPPs, MEPs and megakaryocyte progenitors (Fig.6D). We then analyzed the transcriptional differences between the remaining del(5q) cells of the responder that presented PCR, and those of the NR patient. This analysis identified 1162244 differentially expressed genes (FDR<0.05) per progenitor (Supplementary Fig.4H, Supplementary Data2). Del(5q) cells from the patient in PCR showed statistically significant enrichment in processes and pathways related to protein ubiquitination, proteasomal protein catabolic process, phosphatidylinositol and autophagosome when compared to the NR. Moreover, these cells also exhibited an increased expression of genes involved in erythropoietin signaling when compared to the cells from the NR (Fig.7A, third panel). Interestingly, these processes are similar to the ones detected for non-del(5q) cells when comparing these cells to those at diagnosis (see previous section), and have been described as a lenalidomide response in non-del(5q) MDS patients47,48. The remaining del(5q) cells from the patient in PCR also exhibited enrichment of PD-L1 expression and PD-1 checkpoint pathway when compared to the refractory patient. These analyses suggested low transcriptional alterations promoted by lenalidomide treatment in the NR patient. Accordingly, DE analysis of del(5q) cells at diagnosis and after treatment in the NR patient yielded 20121 differentially expressed genes per hematopoietic progenitor (Supplementary Fig.4I, Supplementary Data2). These few differences resulted in subtle changes in protein ubiquitination and cell cycle-related processes after treatment (Supplementary Fig.6B, Supplementary Table4), showcasing that lenalidomide did not have a high transcriptional impact on del(5q) cells of the NR.

GRN analysis demonstrated a large number of regulons that showed changes in activity after treatment in del(5q) cells from the patient in the PCR but not in the refractory patient. For example, regulons driven by IRF1, JARID2, NCOR1, and CUX1, which showed aberrant low activity at diagnosis that was partially recovered upon treatment, presented very reduced activity in the NR patient, which was lower than that observed in the PCR, and at diagnosis (Fig.7D). Collectively, these results suggest that inNR patients, lenalidomide treatment is not able to reverse part of the transcriptional lesions carried by (5q) cells, which seems to be associated with the lack of hematological response.

Link:
Single-cell transcriptional profile of CD34+ hematopoietic progenitor cells from del(5q) myelodysplastic syndromes and ... - Nature.com

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Stem Cells Market Expected to Expand at a Steady 2024-2031 | - openPR

Former two-weight champion, Conor McGregor has revealed he underwent treatment to address his toe fractured which ruled him from UFC 303 this weekend, through the use of stem cells which he has now theorized may have worsened his injury.

McGregor, a former undisputed lightweight champion and featherweight titleholder, has been sidelined for the last three years from the Octagon suffering a fractured left tibia and fibula in a first round doctors stoppage TKO loss to former interim gold holder, Dustin Poirier.

And slated to make his return at UFC 303 next weekend during International Fight Week, McGregor withdrew from his welterweight fight with Michael Chandler, citing a gut-wrenching fractured toe which is expected to sideline him for a potential period of two months.

Already planning his comeback as he eyes an August return to action, ex-two-weight kingpin, McGregor revealed he underwent stem cell treatment to address his toe injury, however, admits hes not sure it has worked to the best of its ability.

They put stem cells Ive done everything that they asked they put stem cells into me, took it from my back and put it in my foot, 20mg (milligrams) from my own back from the bone marrow in, Conor McGregor told Severe MMA. Right into the [toe] break.

But my f*cking toe is sore, mate, Conor McGregor explained. And I dont know if the stem cells into the break was the right move. I dont think its the swelling anymore I think its just the fluids or stem cells in my toe. So, Im like, Am I going to have a f*cking swollen toe all the time now?

Still planning to make a summer outing despite uncertainty from UFC CEO, Dana White, McGregor confirmed he would still chase a fight with the above-mentioned, Chandler in his immediate return to active competition.

Do you think Conor McGregor can fight this year?

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Conor McGregor Fears Stem Cell Treatment Worsened UFC 303 Injury Setback: 'My Toe Is Sore' - LowKick MMA

The mailings promised Life Without Pain! via stem cell injections or IVs administered in a patients own home. The allure was obvious: more than 20% of U.S. adults suffer from chronic pain.

A court exhibit from a lawsuit filed by Iowa Attorney General Brenna Bird is seen on a laptop computer May 8 in Urbandale, Iowa.

The flyers invited Iowans to free dinners across the state. Afterward, sales people traveled to potential customers homes for high-pressure pitches disguised as pre-screenings, according to prosecutors. More than 250 people signed up, paying $3,200 to $20,000 each for a total of $1.5 million. For this, a nurse practitioner came to their homes to administer injections and IVs filled with stem cells derived from umbilical cords.

Yet experts and regulators have alternately labeled such treatments as ripoffs, scams or simply unproven. In some cases, studies have documented real harm.

Last fall, Iowas attorney general sued two proprietors responsible for the mailings in her state, naming a Minnesota man who hosts a Christian entrepreneurship podcast and his Florida business partner for allegedly deceiving consumers, many of them elderly.

In bringing the lawsuit, Iowa joined attorneys general in New York, North Dakota, Georgia, Nebraska, Arkansas and Washington state who have sued businesses alleging they fraudulently promoted unproven stem cell treatments.

Stem cells have long fascinated researchers because of their ability to reproduce and, in some cases, transform into other cell types. Because of this, they are thought to hold the potential for treating many diseases and injuries.

But the FDA has approved only a handful of such therapies, and only for certain forms of blood cancer and immune system disorders. Stem cells are considered experimental for most uses, despite being marketed as a treatment for everything from autism and emphysema to sports injuries.

The FDA has repeatedly warned Americans to be wary of businesses hawking unapproved, unproven and costly stem cell therapies, which occasionally have caused blindness, bacterial infections and tumors.

In a 2020 notice, the agency expressed concern about patients being misled about products that are illegally marketed, have not been shown to be safe or effective, and, in some cases, may have significant safety issues.

Dr. Jeffrey Goldberg, chair of ophthalmology at the Byers Eye Institute at Stanford University, whose work has documented vision loss in some patients treated with cells removed from patients' own bodies, processed and reinjected, lamented that people are "desperately willing to shell out large sums of money for unproven and in some cases, explicitly sort of sham, so-called therapeutics.

Since August 2017, the FDA has issued about 30 warning letters regarding the unproven treatments.

Experts, including Dr. Paul Knoepfler, a stem cell researcher at the University of California at Davis, and Leigh Turner, a bioethicist at the University of California, Irvine, are among those who have raised alarm that such federal action is too little to regulate a U.S. industry which Turner estimated in 2021 topped 2,700 clinics.

Because states can seek substantial fines against wayward operators, Turner said their legal actions offer promise.

"If you look at them collectively, they might over time start to have an impact, he said.

The FDA offers training to attorneys general pursuing such cases. Dr. Peter Marks, director of the FDAs Center for Biologics Evaluation and Research, said federal regulators partner with state law enforcers in a shared mission.

Iowa Attorney General Brenna Bird speaks during a town hall campaign event for Republican presidential candidate Nikki Haley on May 17, 2023, in Ankeny, Iowa.

That puts people like Iowa Attorney General Brenna Bird on the front lines.

Last year, Bird brought the case over mailers offering Iowans a pain-free life, naming the now dissolved Biologics Health and Summit Partners Group, which operated under the name Summit Health Centers, as defendants. The state also sued the companies' proprietors: Rylee Meek, of Prior Lake, Minnesota, and Scott Thomas, of Thonotosassa, Florida.

Neither man claims to have any medical training. Yet over a series of free dinners across Iowa, attendees listened to their presentations about how stem cells could ostensibly repair damage linked to back or joint pain. The claims came despite an FDA warning that no such product has been approved to treat any orthopedic condition.

One testimonial featured a woman quoted as saying she had multiple sclerosis, fibromyalgia, degenerative joint problems and scoliosis. It implied the treatment worked so well she was able to stop using a walker and taking opioids. Prosecutors say that left people believing stem cells are effective at treating all the conditions listed.

The company offered packages ranging from 5 million cells to up to 60 million to fix customers' ailments. Iowas lawsuit described the practices as scattershot, for-profit experimentations.

Research has shown dead cells are often injected, Knoepfler said.

The Iowa case is still in the discovery stage, with the trial set for March 2025.

Meek and Thomas did not return multiple text and email messages from The Associated Press. Nor did their attorney, Nathan Russell, though he did rebut many of the allegations in court filings, including that the promotional information was deceptive or misleading. The filing stressed that Meek and Thomas always emphasized they were not doctors.

Instead, Meek promoted himself as the $100 million man and touted his business prowess on his Kings Council podcast. His and Thomas book, Intentional Influence in Sales: The Power of Persuasion with Neuro-linguistic Programming, is described as a way to get people to think the way you want them to think, without them even realizing it.

Nearly a quarter of Americans struggle with symptoms of depression, according to the latest Centers for Disease Control and Prevention data from an October 2023 survey. That number is down from 2020 to 2021, when the COVID-19 pandemic exacerbated mental health conditions for millions of Americans.

Like other forms of mental illness, depression impacts groups of people differently depending on their unique backgrounds and experiences. While depression is among the most common forms of mental illness, some portions of the U.S. are seeing rates of depression fall faster than others.

Northwell Health partnered with Stacker to look at which groups of people are the most likely to feel depressed, using data from the CDC.

Signs someone may have depression include an inability to focus, thoughts of death or suicide, hopelessness, and low self-worth, as well as changes in appetite and sleep patterns, according to the World Health Organization.

Depression can be transitorybrought on by the loss of a loved one or other difficult life eventsor chronic, such as for those who live with bipolar disorder. The latest data on depression rates suggest some of the uptick in depression during COVID-19 may have been more of the former.

Depression has lingered at elevated levels for some communities, including young people and those who identify as part of the LGBTQ+ community.

Americans ages 18 to 29 years old report the highest levels of depression, with those 30 to 49 years old showing the next highest levels, according to the CDC. Rates of depression taper off even more as Americans clear the age of 60.

Higher reported rates of depression in young people could partially be attributed to the way each generation views mental illness. Members of Gen Z, those born between 1997 and 2012, have been more open to talking about mental illness and seeking therapy, for example, than older generations who came of age at a time when mental health disorders were heavily stigmatized in media and popular culture.

Surveys have found that discrimination is often cited as a significant source of stress; Black and Hispanic adults, specifically, report higher levels of stress from discrimination compared to their white peers.

When it comes to depression rates, a similar trend appears. Hispanic, multiracial, and Black Americans report elevated rates of depression compared to white Americans, according to the latest survey data the CDC collected in late 2023.

Furthermore, LGBTQ+ Americans have reported higher levels of stress and mental illness compared to straight, cisgender people. Transgender individuals are also more than six times as likely to attempt suicide, according to a Swedish study published in The American Journal of Psychiatryone of the only studies to compile such data for an entire country over a 10-year period.

The current rates of depression among more vulnerable groups are particularly concerning at a time when mental health professionals are struggling to meet a higher demand for mental health care services.

Story editing byShannon Luders-Manuel. Copy editing by Tim Bruns.

This story originally appeared on Northwell Health and was produced and distributed in partnership with Stacker Studio.

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