Expansion Of Stem Cells Research Articles

Abstract SY07-01: p53-mediated stem cell competition: Insights into mechanisms of clonal dominance

Abstract TP53 Mutations confer clonal advantage in several tissues contributing to tumorigenesis. It has traditionally been proposed that heightened resistance to apoptosis/cell cycle arrest can account for clonal advantage. P53 mediated clonal dominance has also been observed in cultured embryonic stem (ES) cells, though it is not clear how this is established. Using live cell imaging we show that human ES cells (hESCs) mutant for TP53 induce death of hES-WT cells and outcompete them. Competition is mediated by apoptosis induction and requires direct cell contact, as in co-cultures of hES-P53KO and hES-WT cells where contact is prevented competition is not observed. To ask if hES-P53KO competition is mediated by mechanical interactions, we cultured cells on deformable membranes to measure their resistance to cell compaction. Interestingly, while hES-WT cells displayed apoptosis induction upon cell compaction, hES-P53KO cells were resistant to compaction-induced apoptosis. We suggest that differential sensitivity to compaction in crowded cultures/tissues can drive p53-induced competitive cell elimination. Identifying the mechanisms by which p53 confers resistance to cell compaction could lead to novel actionable targets to prevent the expansion of p53 mutant stem cells in cancerous and pre-cancerous lesions. Citation Format: Eugenia Piddini. p53-mediated stem cell competition: Insights into mechanisms of clonal dominance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY07-01.

Synergistic effect and molecular mechanism of nicotinamide and UM171 in ex vivo expansion of long-term hematopoietic stem cells

IntroductionSeveral approaches to expand human hematopoietic stem cells (HSCs) have been reported, but the ability of these methods to expand long-term hematopoietic stem cells (LT-HSCs) remains to be improved, which limits the application of HSCs-based therapies. MethodsCD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and then cultured for 12 d in a serum-free medium. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Colony-forming cell (CFC) assays can evaluate multi-lineage differentiation potential of HSCs. Real-time polymerase chain reaction was employed to detect the expression of genes related to self-renewal programs and antioxidant activity. DCFH-DA probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array. ResultsHere, we show a combination, Nicotinamide (NAM) combined with pyrimidoindole derivative UM171, can massively expand LT-HSCs ex vivo, and the expanded cells maintained the capability of self-renewal and multilineage differentiation. Additionally, our data indicated that UM171 promoted self-renewal of HSCs by inducing HSCs entry into the cell cycle and activating Notch and Wnt pathways, but the infinite occurrence of this process may lead to mitochondrial metabolism disorder and differentiation of HSCs. NAM kept HSCs in their primitive and dormant states by reducing intracellular ROS levels and upregulating the expression of stemness related genes, so we believed that NAM can act as a brake to control the above process. ConclusionsThe discovery of the synergistic effect of NAM and UM171 for expanding LT-HSCs provides a new strategy in solving the clinical issue of limited numbers of HSCs.

Abstract 981: Robustness of fragmentation-based cell-free DNA approaches to clonal hematopoiesis

Abstract INTRODUCTION: Clonal hematopoiesis (CH) is the result of the clonal expansion of hematopoietic stem cells which acquire and pass on somatically gained mutations, including SNVs as well as arm level losses and gains, defined here as mosaic deletions and amplifications (mDAs). The proportion of blood cells in circulation containing CH alterations increases with age and environmental exposures such as smoking, leading to a higher prevalence in many high-risk cancer screening populations. CH remains a major confounder for mutation-based liquid biopsy tests evaluating circulating cell-free DNA (cfDNA), since these assays are unable to discriminate between mutations originating from the tumor as opposed to CH. While the impact of CH mutations on fragmentation-based methods of detecting ctDNA appears to be limited, the effect of mDAs has not yet been explored. METHODS: To assess the impact of mDAs on screening efforts using whole genome sequencing (WGS) of cfDNA, we performed copy number analyses on cfDNA from plasma collected from 895 individuals consisting of high-risk individuals meeting the USPSTF guidelines for annual lung cancer screening using low-dose computed tomography. From a subset of 203 available donors (157 without cancer and 46 with lung cancer), matched genomic DNA from white blood cells (WBCs) were extracted from the isolated buffy coat and WGS was performed to evaluate whether ploidy in cfDNA plasma is derived from WBCs. Genome-wide fragmentation features (short to long ratios and fragment size densities), coverage-based statistics, and copy number (CN) statistics adjusting for mDAs were calculated (CNadj). Machine learning models to calculate the probability of cancer based on these features were then derived. RESULTS: 3.8% (95% CI 0.9%-6.8%) of individuals without cancer had an mDA in their WBCs that were also observed in the plasma (p < 0.01, permutation test). Fragmentation features were not different in regions with mDAs in individuals without cancer (p = 0.619; permutation test) but were altered in regions of tumor-derived chromosomal changes in patients with cancer (p < 0.01; permutation test). Coverage-based statistics were impacted by mDAs (p < 0.01; Wilcoxon test). Machine learning models fit with CNadj showed improved performance over CN. However, machine learning models fit with fragmentation features compensated for this difference overall showing robustness to mDAs (p = 0.45; bootstrap) demonstrating the robustness of cfDNA fragmentomes for detecting lung cancer. CONCLUSIONS: While coverage-based statistics of WGS analyses of cfDNA may be affected by mDAs, fragmentation-based approaches relying on cfDNA fragment length are robust to effects of CH. CNadj is an improved statistic for detecting tumor-derived chromosomal copy number changes in cfDNA, providing increased specificity by conditioning on fragment size distributions. Citation Format: Stephen Cristiano, Bahar Alipanahi, Jamie E. Medina, Lorenzo Rinaldi, Nicholas Dracopoli, Robert B. Scharpf, Alessandro Leal, Victor E. Velculescu, Jennifer Tom. Robustness of fragmentation-based cell-free DNA approaches to clonal hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 981.

Abstract 4398: TET2 accelerates clonal hematopoiesis in an age-dependent manner

Abstract Aging is a process of systemic deterioration and the most significant risk factor for cancers. Clonal hematopoiesis (CH) commonly occurs with aging and links to higher mortality, leukemia risk, and cardiovascular diseases. Age-related CH involves the abnormal clonal expansion of hematopoietic stem cells (HSCs) bearing somatic mutations in genes frequently mutated in leukemia, including genes encoding epigenetic regulators, such as the DNA demethylase TET2. While such mutations are known to alter the HSC epigenome, the mechanisms through which these mutations drive HSC self-renewal, myeloid transformation, and inflammatory response remain elusive. In this study, we hypothesize that aging and CH mutations cooperatively reshape the HSC transcriptomic landscape and enhancing HSC competitive advantage that facilitates clonal expansion. Recently, TET2-mutant CH in humans has been strongly associated with epigenetic age acceleration. Using single-cell multi-omic analyses and flow-cytometric phenotyping, we demonstrated, for the first time, that HSC aging processes at transcriptomic, epigenomic, and cellular levels are accelerated by Tet2 deficiency in mice, in an age-dependent manner. Tet2-deficient HSCs increase the HSC aging scores at an older age of greater than or equal to 16 months, but not at a young or mid-age, based on gene expression and chromatin accessibility at a single-cell resolution. This age acceleration was further validated by epigenetic clock analysis of DNA methylation mapping. Moreover, we found that Tet2 deficiency drives a significant expansion of HSCs, a cellular hallmark of HSC aging, in an age-dependent manner. Specifically, we observed increased expression of S100a6, Cdk6, Egr1 in Tet2-deficient HSCs from older mice, suggesting that Tet2 deficiency hijacks the HSC expansion strategy during HSC aging by enhanced self-renewal. We also observed that the differentially expressed genes between Tet2-deficient older HSCs and control older HSCs are associated with open chromatin accessibility. Many of these genes are enriched in the targets of specific transcription factors (TFs), such as Egr1, Creb1, Stat family and Hoxb8. These target genes play critical roles in inflammatory pathways and HSC functions. In conclusion, our findings reveal that Tet2 deficiency significantly contributes to the acceleration of HSC aging, and hijacks the HSC expansion strategy (e.g., self-renewal) during HSC aging, which exacerbates age-related clonal hematopoiesis. By elucidating the transcriptomic and epigenomic alterations in Tet2-deficient HSCs, our study provides novel insights into how age and somatic mutations collaborate to promote the pathogenesis of age-related hematological diseases. These discoveries not only enhance our understanding of HSC aging mechanisms but also offer potential biomarkers for early detection and targets for therapeutic intervention in age-associated clonal disorders. Citation Format: Shilpita Karmakar, Yang Liu, Efthymios Motakis, Kaustav Sengupta, Travis Roeder, Kristina Mujica, Jennifer Trowbridge, Hideyuki Oguro, Sheng Li. TET2 accelerates clonal hematopoiesis in an age-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4398.

Abstract 281: The function of FGFR2+ cancer-associated fibroblasts on cancer stem cell expansion of esophageal squamous cell carcinoma

Abstract Aims: FGFR2+ fibroblasts were reported to promote tumor progression, but the mechanisms remained unexplored in esophageal squamous cell carcinoma (ESCC).Methods: Multiplex immunohistochemistry (mIHC) displayed the distributions of FGFR2+ fibroblasts and cancer stem cells (CSCs). After FGFR2+ fibroblast isolation, tumor cells were stimulated with fibroblasts supernatant to assess radioresistance and CSC properties. RNA-seq analysis identified the downstream BMP5 expression.Results: In our preliminary study, the immunohistochemistry results showed that FGFR2+ fibroblasts were always represented in the area near cancer stem cells (CSCs), and FGFR2+ fibroblasts infiltration was associated with poor outcomes. When we cocultured FGFR2+ fibroblasts with tumor cells, we found the proportion of CSCs were upregulated. The results of RNA sequencing and ELISA showed that FGFR2+ fibroblasts overexpressed BMP5. Using FGFR2+ fibroblasts or BMP5 to stimulate cancer cells, the proportion of cancer stem cells was increased, the capabilities of sphere formation and survival after radiation were enhanced. Both RNA sequencing and immunoblotting results indicated that BMP5 affected H2AX phosphorylation in DNA repair pathway and activated its potential upstream pathway MAPK signaling pathway. In vivo experiments also showed that FGFR2+ fibroblasts could upregulate CSCs proportions.Conclusions: Our findings suggested that BMP5 derived from FGFR2+ fibroblasts might induce CSCs expansion and led to poor prognosis in the ESCC. Citation Format: Ying Wang, Ping Feng, Tong-chao Jiang, Xin-yuan Guan, Yun-fei Xia. The function of FGFR2+ cancer-associated fibroblasts on cancer stem cell expansion of esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 281.

An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

TMEM120B strengthens breast cancer cell stemness and accelerates chemotherapy resistance via β1-integrin/FAK-TAZ-mTOR signaling axis by binding to MYH9

BackgroundBreast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however, the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues, especially in malignant tissues; however, its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance.MethodsBoth bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung, breast, gastric, colon, and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next, colony formation assay, MTT assay, EdU assay, transwell assay, wound healing assay, flow cytometric analysis, sphere formation assay, western blotting analysis, mouse xenograft model analysis, RNA-sequencing assay, immunofluorescence assay, and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation, invasion, stemness, chemotherapy sensitivity, and integrin/FAK/TAZ/mTOR activation. Further, liquid chromatography–tandem mass spectrometry analysis, GST pull-down assay, and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B, myosin heavy chain 9 (MYH9), and CUL9.ResultsTMEM120B expression was elevated in lung, breast, gastric, colon, and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage, lymph node metastasis, and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation, invasion, and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9, which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore, TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely, overexpression of TMEM120B-∆CCD delayed the formation of FAs, suppressed TAZ-mTOR signaling, and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1–2) than in those with better outcomes (Miller/Payne grades 3–5).ConclusionsOur study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the β1-integrin/FAK-TAZ-mTOR signaling axis, maintaining stemness and accelerating chemotherapy resistance.

Hematopoietic stem cells with granulo-monocytic differentiation state overcome venetoclax sensitivity in patients with myelodysplastic syndromes

The molecular mechanisms of venetoclax-based therapy failure in patients with acute myeloid leukemia were recently clarified, but the mechanisms by which patients with myelodysplastic syndromes (MDS) acquire secondary resistance to venetoclax after an initial response remain to be elucidated. Here, we show an expansion of MDS hematopoietic stem cells (HSCs) with a granulo-monocytic-biased transcriptional differentiation state in MDS patients who initially responded to venetoclax but eventually relapsed. While MDS HSCs in an undifferentiated cellular state are sensitive to venetoclax treatment, differentiation towards a granulo-monocytic-biased transcriptional state, through the acquisition or expansion of clones with STAG2 or RUNX1 mutations, affects HSCs’ survival dependence from BCL2-mediated anti-apoptotic pathways to TNFα-induced pro-survival NF-κB signaling and drives resistance to venetoclax-mediated cytotoxicity. Our findings reveal how hematopoietic stem and progenitor cell (HSPC) can eventually overcome therapy-induced depletion and underscore the importance of using close molecular monitoring to prevent HSPC hierarchical change in MDS patients enrolled in clinical trials of venetoclax.

Low-Dose Sorafenib Promotes Cancer Stem Cell Expansion and Accelerated Tumor Progression in Soft Tissue Sarcomas.

The cancer stem cell (CSC) hypothesis postulates that heterogeneous human cancers harbor a population of stem-like cells which are resistant to cytotoxic therapies, thus providing a reservoir of relapse following conventional therapies like chemotherapy and radiation (RT). CSCs have been observed in multiple human cancers, and their presence has been correlated with worse clinical outcomes. Here, we sought to evaluate the impact of drug dosing of the multi-tyrosine kinase inhibitor, sorafenib, on CSC and non-CSCs in soft tissue sarcoma (STS) models, hypothesizing differential effects of sorafenib based on dose and target cell population. In vitro, human cancer cell lines and primary STS from surgical specimens were exposed to escalating doses of sorafenib to determine cell viability and expression of CSC marker aldehyde dehydrogenase (ALDH). In vivo, ALDHbright CSCs were isolated, exposed to sorafenib, and xenograft growth and survival analyses were performed. We observed that sarcoma CSCs appear to paradoxically respond to the tyrosine kinase inhibitor sorafenib at low doses with increased proliferation and stem-like function of CSCs, whereas anti-viability effects dominated at higher doses. Importantly, STS patients receiving neoadjuvant sorafenib and RT on a clinical trial (NCT00864032) showed increased CSCs post therapy, and higher ALDH scores post therapy were associated with worse metastasis-free survival. These data suggest that low-dose sorafenib may promote the CSC phenotype in STS with clinically significant effects, including increased tumor growth and higher rates of metastasis formation in sarcoma patients.

Postbiotics from Lactobacillus delbrueckii Alleviate Intestinal Inflammation by Promoting the Expansion of Intestinal Stem Cells in S. Typhimurium-Induced Mice.

Previous studies have demonstrated that L. delbrueckii plays beneficial roles in modulating the gut microbiota, enhancing the intestinal barrier, and promoting animal growth. Postbiotics have a similar or even superior effect in protecting intestinal health compared to probiotics due to their excellent stability, extended shelf life, and safety. However, the protective effects and underlying mechanism of postbiotics from L. delbrueckii in intestinal inflammation remain unclear. In this study, we demonstrated the beneficial impact of postbiotics from L. delbrueckii on intestinal health by establishing a S. Typhimurium-induced intestinal inflammation model in mice, which included inactivated bacteria and supernatant. The results revealed that the probiotics and postbiotics from L. delbrueckii increased the survival rate and body weight of S. Typhimurium-induced mice, increased the level of IL-10, and decreased the levels of TNF-α and IL-6, thereby alleviating intestinal inflammation. Meanwhile, treatment with postbiotics decreased the levels of D-LA, DAO, and LPS and promoted the expression of Occludin, ZO-1, and Claudin-1 in the serum and jejunum, suggesting an improvement in intestinal barrier function by postbiotics. Additionally, the postbiotics modulated gut microbial diversity, increased the ratio of Firmicutes and Bacteroidetes, and restored the abundance of Muribaculaceae, Lachnospiraceae_NK4a136_groups, and Alloprevotella in S. Typhimurium-infected mice. Moreover, postbiotics from L. delbrueckii promoted the expansion of intestinal stem cells (ISCs) and increased the numbers of Paneth and Goblet cells. Taken together, these data revealed the beneficial role of postbiotics from L. delbrueckii in protecting against intestinal inflammation by promoting the expansion of ISCs.

Improving hematopoietic differentiation from human induced pluripotent stem cells by the modulation of Hippo signaling with a diarylheptanoid derivative

BackgroundThe diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown.MethodWe utilized a defined cocktail of cytokines without serum or feeder followed by the supplementation of ASPP 049 to produce hematopoietic stem/progenitor cells (HSPCs). Flow cytometry and trypan blue exclusion analysis were used to identify nonadherent and adherent cells. Nonadherent cells were harvested to investigate the effect of ASPP 049 on multipotency using LTC-IC and CFU assays. Subsequently, the mechanism of action was explored through transcriptomic profiles, which were validated by qRT-PCR, immunoblotting, and immunofluorescence analysis.ResultThe supplementation of ASPP 049 increased the number of phenotypically defined primitive HSPCs (CD34+CD45+CD90+) two-fold relative to seeded hiPSC colonies, indicating enhanced HSC derivation from hiPSCs. Under ASPP 049-supplemented conditions, we observed elevated HSPC niches, including CD144+CD73− hemogenic- and CD144+CD73+ vascular-endothelial progenitors, during HSC differentiation. Moreover, harvested ASPP 049-treated cells exhibited improved self-renewal and a significantly larger proportion of different blood cell colonies with unbiased lineages, indicating enhanced HSC stemness properties. Transcriptomics and KEGG analysis of sorted CD34+CD45+ cells-related mRNA profiles revealed that the Hippo signaling pathway is the most significant in responding to WWTR1/TAZ, which correlates with the validation of the protein expression. Interestingly, ASPP 049-supplemented HSPCs upregulated 11 genes similarly to umbilical cord blood-derived HSPCs.ConclusionThese findings suggest that ASPP 049 can improve HSC-generating protocols with proliferative potentials, self-renewal ability, unbiased differentiation, and a definable mechanism of action for the clinical perspective of hematopoietic regenerative medicine.Graphical abstract

Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

In vivo and in vitro effects of cord blood hematopoietic stem and progenitor cell (HSPC) expansion using valproic acid and/or nicotinamide

BackgroundHigh self-renewal capacity and most permissive nature of umbilical cord blood (CB) results with successful transplant outcomes but low hematopoietic stem and progenitor cell (HSPC) counts limits wider use. In order to overcome this problem ex vivo expansion with small molecules such as Valproic acid (VPA) or Nicotinamide (NAM) have been shown to be effective. To the best of our knowledge, the combinatory effects of VPA and NAM on HSPC expansion has not been studied earlier. The aim of this study was to analyze ex vivo and in vivo efficacy of VPA and NAM either alone or in combination in terms of expansion and engraftment. MethodsA total of 44 CB units were included in this study. To determine the ex vivo and in vivo efficacy, human CB CD34+ cells were expanded with VPA and/or NAM and colony forming unit (CFU) assay was performed on expanded HSPC. Xenotransplantation was performed simultaneously by intravenous injection of expanded HSPC to NOD-SCID gamma (NSG) mice (n = 22). Significance of the difference between the expansion groups or xenotransplantation models was analyzed using t-test, Mann-Whitney, ANOVA or Kruskal-Wallis tests as appropriate considering the normality of distributions and the number of groups analyzed. ResultsIn vitro CD34+ HSPC expansion fold relative to cytokines-only was significantly higher with VPA compared to NAM [2.23 (1.07–5.59) vs 1.48 (1.00–4.40); p < 0.05]. Synergistic effect of VPA+NAM has achieved a maximum relative expansion fold at 21 days (D21) of incubation [2.95 (1.00–11.94)]. There was no significant difference between VPA and VPA+NAM D21 (p = 0.44). Fold number of colony-forming unit granulocyte-macrophage (CFU-GM) colonies relative to the cytokine-only group was in favor of NAM compared to VPA [1.87 (1.00–3.59) vs 1.00 (1.00–1.81); p < 0.01]. VPA+NAM D21 [1.62 (1.00–2.77)] was also superior against VPA (p < 0.05). There was no significant difference between NAM and VPA+NAM D21. Following human CB34+ CB transplantation (CBT) in the mouse model, fastest in vivo leukocyte recovery was observed with VPA+NAM expanded cells (6 ± 2 days) and the highest levels of human CD45 chimerism was detectable with VPA-expanded CBT (VPA: 5.42 % at day 28; NAM: 2.45 % at day 31; VPA+NAM 1.8 % at day 31). ConclusionOur study results suggest using VPA alone, rather than in combination with NAM or NAM alone, to achieve better and faster expansion and engraftment of CB HSPC.

Reconstructing phylogenetic trees from genome-wide somatic mutations in clonal samples.

Phylogenetic trees are a powerful means to display the evolutionary history of species, pathogens and, more recently, individual cells of the human body. Whole-genome sequencing of laser capture microdissections or expanded stem cells has allowed the discovery of somatic mutations in clones, which can be used as natural barcodes to reconstruct the developmental history of individual cells. Here we describe Sequoia, our pipeline to reconstruct lineage trees from clones of normal cells. Candidate somatic mutations are called against the human reference genome and filtered to exclude germline mutations and artifactual variants. These filtered somatic mutations form the basis for phylogeny reconstruction using a maximum parsimony framework. Lastly, we use a maximum likelihood framework to explicitly map mutations to branches in the phylogenetic tree. The resulting phylogenies can then serve as a basis for many subsequent analyses, including investigating embryonic development, tissue dynamics in health and disease, and mutational signatures. Sequoia can be readily applied to any clonal somatic mutation dataset, including single-cell DNA sequencing datasets, using the commands and scripts provided. Moreover, Sequoia is highly flexible and can be easily customized. Typically, the runtime of the core script ranges from minutes to an hour for datasets with a moderate number (50,000-150,000) of variants. Competent bioinformatic skills, including in-depth knowledge of the R programming language, are required. A high-performance computing cluster (one that is capable of running mutation-calling algorithms and other aspects of the analysis at scale) is also required, especially if handling large datasets.

The Use of Human Platelet Lysate as a Coating Substance for Adipose-Derived Stem Cell Expansion.

Large-scale production of mesenchymal stromal cells is essential for sufficient therapeutic doses in regenerative medicine. However, long-term cultivation encounters limited cell growth and cellular aging. Therefore, an alternative cell culture approach that promotes proliferation and attenuates cell senescence is required. Human platelet lysate (HPL) is a potent supplement for in vitro cell expansion. Applying HPL as a coating material can potentially improve mesenchymal stromal cell cultures. To examine the capacity of HPL, it was used to pre-coat a tissue culture plate for in vitro adipose-derived mesenchymal stromal cell expansion. Alterations in biological features of adipose-derived stem cells (ADSCs) were investigated, including cell adhesion assays, cell proliferation, population doubling time, and cellular senescence. ADSCs cultured on HPL-coated plates significantly increased cell adhesion rate, shortened population doubling time, and stimulated cell growth. The senescent cells were significantly decreased in ADSCs cultured in an HPL-coated plate, and the expression levels of senescence-associated genes, including p16, p21, and p53, were downregulated. Furthermore, Western blotting analysis revealed that HPL was enriched with fibronectin and vitronectin, essential cell adhesive proteins. HPL was effectively used as a coating material for ADSC expansions. Cellular cultivation on the HPL coating is an alternative approach for producing mesenchymal stromal cells.

Process Intensification in Human Pluripotent Stem Cell Expansion with Microcarriers

Given the demands human induced pluripotent stem cell (hiPSC)-based therapeutics place on manufacturing, process intensification strategies which rapidly ensure the desired cell quality and quantity should be considered. Within the context of antibody and vaccine manufacturing, one-step inoculation has emerged as an effective strategy for intensifying the upstream process. This study therefore evaluated whether this approach could be applied to the expansion of hiPSCs in flasks under static and in microcarrier-operated stirred bioreactors under dynamic conditions. Our findings demonstrated that high density working cell banks containing hiPSCs at concentrations of up to 100 × 106 cells mL−1 in CryoStor® CS10 did not impair cell growth and quality upon thawing. Furthermore, while cell distribution, growth, and viability were comparable to routinely passaged hiPSCs, those subjected to one-step inoculation and expansion on microcarriers under stirred conditions were characterized by improved attachment efficiency (≈50%) following the first day of cultivation. Accordingly, the process development outlined in this study establishes the foundation for the implementation of this intensified approach at L-scale.

Ex vivo expansion of hematopoietic stem cells in two/ three-dimensional co-cultures with various source of stromal cells

The ex vivo expansion of hematopoietic stem cells, with both high quantities and quality, is considered a paramount issue in cell and gene therapy for hematological diseases. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells reveal the importance of using 2D and 3D coculture as a physiological system simulator in the proliferation, differentiation, and homeostasis of HSCs. Herein, the capacity of mesenchymal stem cells derived from different sources to support the expansion and maintenance of HSPC was compared with each other. We evaluated the fold increase of HSPC, CD34 marker expression, cytokine secretion profile of different MSCs, and the frequency of hematopoietic colony-forming unit parameters. Our results show that there was no significant difference between adipose tissue-MSC, Wharton jelly-MSC, and Endometrial-MSCs in HSPC expansion (fold increase: 34.74±4.38 in Wj-MSC, 32.22±5.07 in AD-MSC, 25.9±1.27 in En-MSCs); However, there were significantly more than the expansion media alone (4.4±0.69). The results obtained from the cytokine secretion analysis also confirm these results. Also, there were significant differences in the clonogenicity of Wj-MSC, En-MSCs, and expansion media (CFU-GEMM: 7±1.73, 2.3±1.15, and 2.3±1.52), which indicated that Wj-MSC could significantly maintain the primitive state. As a result, using Wj-mesenchymal stem cells on a 3D coculture system effectively increases the HSPC expansion and maintains the colonization potential of hematopoietic stem cells.

The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

Pre-mRNA splicing is a precise regulated process, and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs), and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remain largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout zebrafish line, and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells (RPCs) by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 knockout zebrafish line, we discovered that prpf31 knockout zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31 deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.

A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

The impact of age and number of mutations on the size of clonal hematopoiesis

Clonal hematopoiesis (CH) represents the clonal expansion of hematopoietic stem cells and their progeny driven by somatic mutations. Accurate risk assessment of CH is critical for disease prevention and clinical decision-making. The size of CH has been showed to associate with higher disease risk, yet, factors influencing the size of CH are unknown. In addition, the characteristics of CH in long-lived individuals are not well documented. Here, we report an in-depth analysis of CH in longevous (≥90 y old) and common (60~89 y old) elderly groups. Utilizing targeted deep sequencing, we found that the development of CH is closely related to age and the expression of aging biomarkers. The longevous elderly group exhibited a significantly higher incidence of CH and significantly higher frequency of TET2 and ASXL1 mutations, suggesting that certain CH could be beneficial to prolong life. Intriguingly, the size of CH neither correlates significantly to age, in the range of 60 to 110 y old, nor to the expression of aging biomarkers. Instead, we identified a strong correlation between large CH size and the number of mutations per individual. These findings provide a risk assessment biomarker for CH and also suggest that the evolution of the CH is influenced by factor(s) in addition to age.