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opencc-zeroOct 2020View details →

Endocannabinoid signalling in stem cells and cerebral organoids drives differentiation to deep layer projection neurons via CB1 receptors

The endocannabinoid (eCB) system, via cannabinoid CB1 receptor, regulates neurodevelopment by controlling neural progenitor proliferation and neurogenesis. CB1 receptor signalling in vivo drives corticofugal deep layer projection neuron development through the regulation of BCL11B and Satb2 transcription factors. Here, we investigated the role of eCB signalling in mouse pluripotent embryonic stem cell-derived neuronal differentiation. Characterization of the eCB system revealed increased expression of eCB-metabolizing enzymes, eCB ligands and CB1 receptors along neuronal differentiation. CB1 receptor knockdown inhibited neuronal differentiation of deep layer neurons and increased upper layer neuron generation, and this phenotype was rescued by CB1 re-expression. Pharmacological regulation with CB1 receptor agonists or elevation of eCB tone with a monoacylglycerol lipase inhibitor promoted neuronal differentiation of deep layer neurons at the expense of upper layer neurons. Patch-clamp analyses revealed that enhancing cannabinoid signalling facilitated neuronal differentiation and functionality. Noteworthy, incubation with CB1 receptor agonists during human iPSC-derived cerebral organoid formation also promoted the expansion of BCL11B+ neurons. These findings unveil a cell-autonomous role of eCB signalling that, via CB1 receptor, promotes mouse and human deep layer cortical neuron development.

opencc-by-4.0Dec 2020View details →

Data from: A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs and hNPCs. The results of our study build on current evidence that CUL9 may have unique functions in different cell types potentially contributing to the difficulty of identifying CUL9 substrates. Information on data/files: Please first unzip Figure_4_CUL9-IP-LCMSMS-data.zip and Figure_7_iTRAQ-data.zip, then follow the description below. CUL9 immunoprecipitation from whole lysates collected for hPSCs were analyzed using LC/MS-MS. IgG was used as a control to determine proteins specifically enriched in the CUL9 IP. This data correlates to Figure 4 of the associated manuscript. Initial CUL9 immunoprecipitation was performed by VG and analyzed by LC-MS/MS at UNC. Fig4-LCMSMS_IPCUL9_Replicate1_UNC Two more immunoprecipitations were performed by VG and analyzed by LC-MS/MS at Vanderbilt University MSRC Proteomics Core. The first run (replicate 1) was used to produce the STRING figure in the associated manuscript, as well as the volcano plot in Supplemental Figure 8. Two Scaffold files contain raw data and our analysis parameters: <ul> <li>Fig4-LCMSMS_IPCUL9_Replicate2_Vanderbilt</li> <li>Fig4-LCMSMS_LCMSMS_IPCUL9_Replicate3_Vanderbilt</li> </ul> Excel files exported from the above Scaffold files contain the raw data in excel format including spectral counts, peptide counts, protein probability, and detailed raw data as it pertains to each identified protein. See these data sets in the folders below: <ul> <li>Fig4-LCMSMS_IPCUL9_Run2_Vanderbilt_CompleteDataSet</li> <li>Fig4-LCMSMS_IPCUL9_Run3_Vanderbilt_CompleteDataSet</li> </ul> CUL9 KO clones were used to identify proteins increased or decreased compared to parental wild-type cell lines using iTRAQ. This data correlates to Figure 8 of the associated manuscript. Two iTRAQ experiments were performed to identify proteins altered in CUL9 KO iPSCs, neural stem cells (NSCs), and NPCs. Each set of experiments was duplicated – each in a different isogenic CUL9 KO clone (Clone 20 or Clone B) iTRAQ labeling of experiment one is as follows: <ol> <li>Label 115: Parental WT NSCs</li> <li>Label 117: CUL9 KO Clone 20 or B NSCs</li> <li>Label 114: Parental WT NPCs</li> <li>Label 116: CUL9 KO Clone 20 or B NPCs</li> </ol> Files containing raw and analyzed data of experiment one are labeled as follows: <ul> <li>Fig7_Experiment1_Clone 002_compared-to-NSC-WT</li> <li>Fig7_Experiment1_Clone001-compared-to-NSC-WT</li> <li>Fig7_Experiment1_Clone001iPSC_compared-to-iPSCWT</li> <li>Fig7_Experiment1_Clone002_compared-to-iPSCWT</li> </ul> iTRAQ labeling of experiment two is as follows: For Clone 20: <ol> <li>Label 115: Parental WT NSCs</li> <li>Label 117: CUL9 KO Clone 20 or B NSCs</li> <li>Label 114: Parental WT iPSCs</li> <li>Label 116: CUL9 KO Clone 20 or B iPSCs</li> </ol> Files containing raw and analyzed data of experiment two are labeled as follows: <ul> <li>Fig7_Experiment2_Clone001NPC_Compared-to-NPC-WT</li> <li>Fig7_Experiment2_Clone001NPC_Compared-to-NSC-WT</li> <li>Fig7_Experiment2_Clone002NPC_Compared-to-NPC-WT</li> <li>Fig7_Experiment2_Clone002NPC_Compared-to-NSC-WT</li> </ul> Please note that the first tab of each excel document contains the full raw data set before analysis. All other tabs contain analyzed data comparing the proteins identified in two cell lines. Analyzed tabs are labeled to indicate which cell lines are being compared. Please note that the CUL9 isogenic clones Clone 20=Clone#1 and Clone B=Clone#2 as referenced in the associated manuscript. Detailed information about the protocols used for collection and analysis of this data can be found in the supplemental information within the associated manuscript.

opencc-byDec 2020View details →

Supporting Information of the article "mesenchymal stem cell-derived exosomes protect trabecular meshwork from oxidative stress"

Supporting Information of the article "mesenchymal stem cell-derived exosomes protect trabecular meshwork from oxidative stress".

opencc-zeroJan 2021View details →

Control of the anterior pituitary cell lineage regulator POU1F1 by the stem cell determinant Musashi

The cytokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction and adult onset obesity. Amongst other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1 (also referred to as Pit1) which, in turn, regulates the specification of somatotrope, lactotrope and thyrotrope cell lineages within the anterior pituitary. Leptin's mechanism of action on somatotropes is sex-dependent, with females demonstrating post-transcriptional control of Pou1f1 mRNA translation. In this study, we have investigated the mechanism that regulates Pou1f1 mRNA translational control and report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In FACS-purified female somatotropes, Msi1 mRNA levels and Musashi1 protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatrotropes and immunocytochemistry analyses of these cells confirms that Musashi1 protein is present in the somatotrope population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3' untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals co-associated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

opencc-by-4.0Jun 2021View details →

Rational design and optimization of synthetic gene switches for controlling cell-fate decisions in pluripotent stem cells

Data underlying the figures in the publication “Rational Design and Optimization of Synthetic Gene Switches for Controlling Cell-fate Decisions in Pluripotent Stem Cells”, published in Metabolic Engineering, 2021, 65, 99-110. <a href="https://doi.org/10.1016/j.ymben.2021.03.009">https://doi.org/10.1016/j.ymben.2021.03.009</a> Table of contents: 1. Dataset 1; Excel file with the curated data underlying the figures. Figure 1. Comparative analysis of synthetic gene switches in HEK-293T and hiPSCs. (A) Erythromycin-responsive synthetic gene switch consisting of a constitutively expressed erythromycin-responsive transactivator (pVH620; PmPGK1-E-VP16-pA) and an inducible Nluc expression vector controlled by an operator containing two erythromycin transcription response (ETR) sites (pVH619; OETR2-PCMVmin-2-Nluc-pA), assessed in terms of production of secreted Nluc 24 h after transfection. EM indicates addition of erythromycin (2.0 µg/mL) to the culture medium. (B) Phloretin-responsive synthetic gene switch consisting of a constitutively expressed phloretin-responsive transactivator (pVH655; PmPGK1-TtgR-VP16-pA) and an inducible Nluc expression vector controlled by an operator containing two TtgR binding sites (pVH654; OTtgO2-PCMVmin-2-Nluc-pA), assessed in terms of production of secreted Nluc 24 h after transfection. Phlo indicates addition of phloretin (50 µM) to the culture medium. (C) Vanillic acid-responsive synthetic gene switch consisting of a constitutively expressed vanillic acid-responsive transactivator (pVH238-1; PmPGK1-VanR-VP16-pA) and an inducible Nluc expression vector controlled by an operator containing two VanR binding sites (pVH294-1.3; OVanO2-PCMVmin-2-Nluc-pA), assessed in terms of production of secreted Nluc 24 h after transfection. VA indicates addition of vanillic acid (500 µM) to the culture medium. Figure 2. Rational design and assembly of a vanillic acid-controlled synthetic gene switch in hiPSCs. (B) Combinatorial analysis of constitutive promoters in hiPSCs for the expression of the VanR-VP16 trans-activator (pVH238-1; PmPGK1-VanR-VP16-pA, pVH238-2; PhEF1a-VanR-VP16-pA) and inducible expression cassette minimal promoters (pVH294-1.1; OVanO2-Pmin-Nluc-pA, pVH294-1.3; OVanO2-PCMVmin-2-Nluc-pA, pVH294-1.2; OVanO2-PCMVmin-1-Nluc-pA), assessed in terms of the production of secreted Nluc 24 h after transfection. (C) Tuning the induction level of the inducible expression cassette in hiPSCs by increasing the number of VanR binding sites within the operator (pVH294-1.1; OVanO2-Pmin-Nluc-pA, pVH294-2.1; OVanO4-Pmin-Nluc-pA, pVH294-3.1; OVanO6-Pmin-Nluc-pA), assessed in terms of the production of secreted Nluc 24 h after co-transfection with PmPGK1-driven VanR-VP16 (pVH238-1; PmPGK1-VanR-VP16-pA). (D) Assessment of the structural assembly of the VanR-VP16 transactivator in hiPSCs, comparing direct fusion of VanR and VP16 (pVH238-1; PmPGK1-VanR-VP16-pA), fusion with a short flexible (pVH261; PmPGK1-VanR-FL1-VP16-pA, FL1; GGGGS) or rigid (pVH263; PmPGK1-VanR-RL1-VP16-pA, RL1; EAAAK) linker, and fusion with a longer flexible (pVH262; PmPGK1-VanR-FL2-VP16-pA, FL2; GGGGSGGGGS) or rigid (pVH264; PmPGK1-VanR-RL2-VP16-pA, RL2; EAAAKEAAAK) linker, assessed in terms of the production of secreted Nluc 24 h after co-transfection with a Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-2.1; OVanO4-Pmin-Nluc-pA). (E) Tuning the induction level of the VA-controlled synthetic gene switch in hiPSCs by promoting active nuclear translocation of VanR-VP16 (pVH238-1; PmPGK1-VanR-VP16-pA) with importin-alpha (pVH250; PmPGK1-NLSSV40-VanR-VP16-pA) or importin-beta (pVH252; PmPGK1-NLSG46-VanR-VP16-pA)-recognized NLS peptide, assessed in terms of the production of secreted Nluc 24 h after co-transfection with a Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-2.1; OVanO4-Pmin-Nluc-pA). (F) Combinatorial analysis for the final assembly of the VA-controlled synthetic gene switch in hiPSCs using a Pmin-driven reporter construct controlled by either four (pVH294-2.1; OVanO4-Pmin-Nluc-pA) or six (pVH294-3.1; OVanO4-Pmin-Nluc-pA) VanR binding sites and different combinations of selected components from previous characterization steps to assemble the transactivator (pVH238-1; PmPGK1-VanR-VP16-pA, pVH263; PmPGK1-VanR-RL1-VP16-pA, pVH262; PmPGK1-VanR-FL2-VP16-pA, pVH250; PmPGK1-NLSSV40-VanR-VP16-pA, pVH252; PmPGK1-NLSG46-VanR-VP16-pA, pVH629; PmPGK1-NLSSV40-VanR-RL1-VP16-pA, pVH628; PmPGK1-NLSSV40-VanR-FL2-VP16-pA, pVH300; PmPGK1-NLSG46-VanR-RL1-VP16-pA, pVH624; PmPGK1-NLSG46-VanR-FL2-VP16-pA), assessed in terms of the production of secreted Nluc 24 h after transfection. (G) Dose-response curves in hiPSCs of the PmPGK1-driven NLSG46-VanR-VP16 (pVH252; PmPGK1-NLSG46-VanR-VP16-pA) and the basic VanR-VP16 (pVH238-1; PmPGK1- VanR-VP16-pA) fusion constructs using the Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-1.2; OVanO4-Pmin-Nluc-pA), assessed in terms of the production of secreted Nluc 24 h after transfection. (B-F) VA indicates addition of vanillic acid (500 µM) to the culture medium. Figure 3. Regulation of the VanA system during the generation of the three germ layers. hiPSCs transiently transfected with the complete VA-controlled expression vector (pVH438; 5’ITR-OVanO4-Pmin-Nluc-pA-PmPGK1-NLSG46-VanR-VP16-2A-NeoR-pA-3’ITR) were specified for 3 days into (A) DE or (B) ME, and for 5 days into (C) EC. The VanA system was maintained in its repressed state by the addition of VA (500 µM) and induced by the removal of VA from the differentiation medium on the last day of differentiation. Induction of the VanA system was assessed in terms of the intracellular production of non-secreted Nluc. Figure 4. Synthetic gene-switch-guided posterior foregut lineage specification. (C) The mRNA expression levels of HHEX and TGIF2 in iHHEX/iTGIF2 hiPSCs cultured for 24 h in the presence of different combinations of VA and DOX were determined by means of qRT-PCR. VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Values were normalized to those in the VA (+)/DOX(-) condition. (G-H) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiated towards PG with FGF7 (25 ng/mL) for 3 days, and subsequently towards PFG with FGF7 (25 ng/mL), noggin (50 ng/mL), and retinoic acid (RA; 2 µM) for 1 day. During differentiation HHEX and TGIF2 was maintained uninduced/repressed (VA on D0-7, No DOX), or HHEX was induced on day 2-3 by the addition of DOX and TGIF2 was induced on day 3-7 by removal of VA from the differentiation medium (VA on D3-7, DOX on D2-3). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. (G) Quantification of PDX1-expressing PFG cells. Flow cytometry results quantification of PDX1-expressing PFG cells differentiated using growth-factor-directed (VA on D0-7, No DOX) and TGIF2/HHEX-guided (VA on D0-3, DOX on D2-3) differentiation protocols. (H) Relative mRNA expression levels for PP (PDX1), PFG (SOX9, HNF6), HP (ALB), MHG (CDX2), and AFG (OTX2) genes on day 7 in iHHEX/iTGIF2 hiPSC-derived PFG cells generated with the indicated HHEX and TGIF2 induction conditions were determined by means of qRT-PCR. Values were normalized to those in day 0 iHHEX/iTGIF2 hiPSCs. Figure S1. Effect of constitutive promoter expression strength (pVH238-1; PmPGK1-VanR-VP16-pA, pVH238-2; PhEF1a-VanR-VP16-pA) on the basal expression levels of inducible vectors containing different minimal promoters (pVH294-1.1; OVanO2-Pmin-Nluc-pA, pVH294-1.3; OVanO2-PCMVmin-2-Nluc-pA, pVH294-1.2; OVanO2-PCMVmin-1-Nluc-pA) relative to mock transfection (pColaDuet-1) in hiPSCs, assessed in terms of the production of secreted Nluc 24 h after transfection. VA indicates addition of vanillic acid (500 µM) to the culture medium. Figure S2. Assessment of optimal fusion configurations for NLS peptides. (A) NLSSV40 or (B) NLSG46 was fused to the N-terminus of VanR-VP16 (pVH250; PmPGK1-NLSSV40VanR-VP16-pA, pVH252; PmPGK1-NLSG46-VanR-VP16-pA), in between VanR and VP16 as a linker (pVH260; PmPGK1-VanR-NLSSV40-VP16-pA, pVH622; PmPGK1-VanR-NLSG46-VP16-pA), or to the C-terminus (pVH251; PmPGK1-VanR-VP16-NLSSV40-pA, pVH623; PmPGK1-VanR-VP16-NLSG46-pA) to identify the best conformation in hiPSCs, assessed in terms of the production of secreted Nluc 24 h after co-transfection with a Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-2.1; OVanO4-Pmin-Nluc-pA). (A-B) VA indicates addition of vanillic acid (500 µM) to the culture medium. Figure S3. Complete expression vector design for stable integration encoding OVanO4-Pmin-driven Nluc expression cassette and PmPGK1-driven NLSG46-VanR-VP16 (pVH438; 5’ITR-OVanO4-Pmin-Nluc-pA-PmPGK1-NLSG46-VanR-VP16-2A-NeoR-pA-3’ITR). (B) Transient performance characterization in hiPSCs assessed in terms of the production of intracellular Nluc 24 h after transfection. VA indicates addition of vanillic acid (500 µM) to the culture medium. Figure S4. Generation of the three germ layers from hiPSCs transfected with complete VA-controlled expression vector (pVH438; 5’ITR-OVanO4-Pmin-Nluc-pA-PmPGK1-NLSG46-VanR-VP16-2A-NeoR-pA-3’ITR). (A) Relative mRNA expression levels of definitive endoderm (DE) genes (EOMES, SOX17, FOXA2, CXCR4) in cells transfected with the complete VA system expression vector after 3 days of DE specification with VA. (B) Relative mRNA expression levels of mesoderm (ME) genes (T, PDGFRa, TBX6, HAND1) in cells transfected with the complete VA system expression vector after 3 days of ME specification with VA. (C) Relative mRNA expression levels of ectoderm (EC) genes (PAX6, NES, OTX2, SOX10) in cells transfected with the complete VA system expression vector after 5 days of EC specification with VA. (A-C) Values were normalized to those of day 0 hiPSCs. VA indicates the addition of vanillic acid (500 µM) to the differentiation medium. Figure S5. Performance comparison and multiplexing of the VanA and rtTA systems in hIPSCs. (A) Comparison of selected inducible expression cassette containing four VanR response elements (pVH294-2.1; OVanO4-Pmin-Nluc-pA) in combination with PmPGK1-driven NLSG46-VanR-VP16 (pVH252; PmPGK1-NLSG46-VanR-VP16-pA) fusion construct relative to the DOX-controlled rtTA system (pVH627; OTetO7-Pmin-Nluc-pA / pVH235; PmPGK1-rtTA-pA) in hiPSCs assessed in terms of the production of secreted Nluc 24 h after transfection. (B) Assessment of multiplexing potential of the VA system (pVH425; OVanO4-Pmin-Nluc-pA / pVH252; PmPGK1-NLSG46-VanR-VP16-pA) and the DOX-controlled rtTA system (pVH424; OTetO7-Pmin-Fluc-pA / pVH235; PmPGK1-rtTA-pA) in hiPSCs assessed in terms of the production of intracellular Nluc and Fluc 48 h after transfection. (A-B) VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (1 µg/mL) to the culture medium. Figure S6. (C) iHHEX/iTGIF2 hiPSCs were differentiated towards anterior primitive streak (APS) and DE using the standard combination of exogenous Wnt (GSK3 inhibition CHIR99021 [CHIR]; 3 µM) and TGF-β (Activin A [AA]; 100 ng/mL) induction. During differentiation, TGIF2 was maintained repressed by the addition of VA (VA on D0-3) and HHEX was maintained uninduced (No DOX). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Relative mRNA expression levels for pluripotency markers (POU5F1, NANOG, SOX2), and definitive endoderm (DE) markers (EOMES, SOX17, FOXA2, CXCR4) on day 3 in iHHEX/iTGIF2 hiPSC-derived DE cells were determined by means of qRT-PCR. Values were normalized to those of day 0 iHHEX/iTGIF2 hiPSCs. (E) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiation towards PG for 1 day with FGF7 (25 ng/mL). During PG specification, TGIF2 was induced alone (VA on D0-3, No DOX), HHEX was induced alone (VA on D0-4, No DOX), or TGIF2 and HHEX were both maintained uninduced (VA on D0-4, No DOX) on day 3-4 by removal or addition of VA and DOX as specified. VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. The expression levels for HHEX and TGIF2 on day 4 during PG differentiation of iHHEX/iTGIF2 hiPSC-derived DE cells were determined by means of qRT-PCR. Values were normalized to GAPDH and TBP expression levels in day 4 iHHEX/iTGIF2 hiPSCs. Figure S7. (B) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiated towards PG with FGF7 (25 ng/mL) for 3 days, and subsequently towards PFG with FGF7 (25 ng/mL), noggin (50 ng/mL), and retinoic acid (RA; 2 µM) for 1 day. During differentiation TGIF2 was maintained repressed under all conditions by the addition of VA to the differentiation media on day 0-7 (VA on D0-7). HHEX was either maintained uninduced (No DOX) or induced on day 2-3, 2-4, or 2-5 by the addition of DOX to the differentiation media (DOX on D2-3, D2-4, or D2-5). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Relative mRNA expression levels for PP (PDX1), HP (ALB), and PFG (SOX9, HNF6) genes on day 7 in iTGIF2/iHHEX hiPSC-derived PFG cells generated with the indicated differentiation protocol and HHEX induction conditions were determined by means of qRT-PCR. Values were normalized to those of day 0 iHHEX/iTGIF2 hiPSCs. (D) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiated towards PG with FGF7 (25 ng/mL) for 3 days, and subsequently towards PFG with FGF7 (25 ng/mL), noggin (50 ng/mL), and retinoic acid (RA; 2 µM) for 1 day. During differentiation HHEX was maintained uninduced under all conditions (No DOX). TGIF2 was either maintained uninduced by the addition of VA (VA on D0-7) or induced on day 5-7, 4-7, or 3-7 by removal of VA from the differentiation medium (VA on D0-5, D0-4, or D0-3). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Relative mRNA expression levels for PP (PDX1), HP (ALB), and PFG (SOX9, HNF6) genes on day 7 in iHHEX/iTGIF2 hiPSC-derived PFG cells generated with the indicated differentiation protocol and TGIF2 induction conditions were determined by means of qRT-PCR. Values were normalized to those of day 0 iHHEX/iTGIF2 hiPSCs.

opencc-zeroFeb 2020View details →

A targeted metabolomics-based assay using human induced pluripotent stem cell-derived cardiomyocytes identifies structural and functional cardiotoxicity potential

Implementing screening assays that identify functional and structural cardiotoxicity earlier in the drug development pipeline has the potential to improve safety and the cost and time required to bring new drugs to market. In this study, a metabolic biomarker-based assay was developed that predicts the cardiotoxicity potential of a drug based on changes in the metabolism and viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Assay development and testing was conducted in two phases: (1) biomarker identification and (2) targeted assay development. In the first phase, metabolomic data from hiPSC-CM spent media following exposure to 66 drugs was used to identify biomarkers that identified both functional and structural cardiotoxicants. Four metabolites that represent different metabolic pathways (arachidonic acid, lactic acid, 2'-deoxycytidine, and thymidine) were identified as indicators of cardiotoxicity. In phase two, a targeted, exposure-based biomarker assay was developed that measured these metabolites and hiPSC-CM viability across an eight-point concentration curve. Metabolite-specific predictive thresholds for identifying the cardiotoxicity potential of a drug were established and optimized for balanced accuracy or sensitivity. When predictive thresholds were optimized for balanced accuracy, the assay predicted the cardiotoxicity potential of 81 drugs with 86% balanced accuracy, 83% sensitivity, and 90% specificity. Alternatively, optimizing the thresholds for sensitivity yields a balanced accuracy of 85%, 90% sensitivity, and 79% specificity. This new hiPSC-CM-based assay provides a paradigm that can identify structural and functional cardiotoxic drugs that could be used in conjunction with other endpoints to provide a more comprehensive evaluation of a drug's cardiotoxicity potential.

opencc-by-4.0Feb 2018View details →

Table S1 - Table S7: Integrated meta-omic analyses of the gastrointestinal tract microbiome in patients undergoing allogeneic stem cell transplantation

Table S1. Number of reads per prokaryotic operational taxonomic unit (OTU) and sample from the cohort. Table S2. Number of reads per eukaryotic operational taxonomic unit (OTU) and sample from the cohort. Table S3. Blood cell counts and clinical data from patient A07. Table S4. Number of reads per operational taxonomic unit (OTU) and sample from patient A07. Table S5. Numbers of identified antibiotic resistance genes and total number of genes. Numbers of identified antibiotic resistance genes in relation to total numbers of genes in samples from patient A07 before and after allo-HSCT and from four healthy individuals. Table S6. Statistics of the metagenomic and metatranscriptomic datasets and the co-assembled contigs. Table S7. Antibiotic resistance genes in population-level genomes and their expression in the pre- and post-treatment sample.

opencc-by-4.0Dec 2023View details →

A method to investigate Pathological Variant load of Mitochondrial DNA in single human Mesenchyma stem cells

Mesenchymal stem cells (MSCs) are the parent cells to many cells of the musculoskeletal system including osteoblasts. Previous work has shown that in mouse models mitochondrial DNA (mtDNA) pathological variants lead to dysfunction of the respiratory chain of these osteoblasts and the premature development of age related osteoporosis. An increased rate of respiratory chain deficiency has also been observed in human osteoblasts. mtDNA mutates at an enhanced rate compared to nuclear DNA and has been linked to the ageing process in humans.  We have developed and optimised an experimental pipeline to isolate single MSCs using flow cytometry before performing next generation sequencing to analyse the pathogenic variant load of 13 patients aged 22-88 to assess if pathogenic variants exist in mesenchymal stem cells and if there are age related changes. Across all patients and all cells mtDNA pathological variants were present at similarly low levels. As per previous studies we found increased pathogenic variants within the mtDNA control region (D-Loop). However overall, there was no significant difference in the distribution of variants across the rest of the genome in all patients. Although a higher proportion of non-synonymous variants were seen this was not statistically significant. We have shown that it is possible to isolate and sequence individual MSCs and observe mtDNA pathological variants. This gives a snapshot in time of the variant load for each patient. Due to low sample numbers compared to other studies investigate mtDNA in other cell types, we did not observe any age related increase in pathological variants. However this adds further evidence that clonally expanded, somatic mtDNA variants are common and could contribute to age related disease including osteoporosis.

opencc-by-4.0Mar 2024View details →

Data for Mechanically-driven Stem Cell Separation in Tissues caused by Proliferating Daughter Cells

Contains animations, data, and source code used for the paper "Mechanically-driven Stem Cell Separation in Tissues caused by Proliferating Daughter Cells" by the same authors.  This repository contains all the data and analysis scripts to support the manuscript The file structure is as follows: - "analysis" contains all the analysis scripts - "gfx" and "animations" the figures and supporting animations - "1-SCpair", "2-SCtissue", "3-BDsimulation", "4-tissue-stochdiv", "6-inertia-effects" all the source code and data for the respective models.

opencc-by-4.0Mar 2024View details →

Early and late consequences of damage to the stem cell compartment following SARS-CoV-2 infection

This application has been reviewed in a previous cycle and we would like to thank reviews for very positive comment. However, to our surprise one reviewer stated that this proposal is not based on our original hypothesis. To address this misunderstanding the role of Nlrp3 inflammasome as a trigger of cytokine storms COVID19 was proposed in the literature for a first time by our team (Ratajczak MZ, Kucia M. SARS-CoV-2 infection and overactivation of Nlrp3 inflammasome as a trigger of cytokine "storm" and risk factor for damage of hematopoietic stem cells. Leukemia. 2020 Jul;34(7):1726-1729. doi: 10.1038/s41375-020-0887-9) and subsequently several groups confirmed this hypothesis and highly cited our initial report. COVID19 or SARS-CoV-2 virus is single-stranded RNA virus, whose infection can be asymptomatic or lead to damage of several vital organs and a fatal complication involving “cytokine storm”, which results in uncontrolled hyperactivation of the immune response by innate immunity cells. The major concern is that we still cannot foresee late complications of this infection including direct or indirect effects on stem cell compartment. SARS-CoV-2 may enter human cells after binding to the angiotensin-converting enzyme 2 (ACE2) receptor and utilizes its surface spike protein (SP) for attachment and entry into the target cells. It has been demonstrated that ACE2 receptor is highly expressed on hematopoietic stem cells (HSCs) and endothelial progenitors (EPCs) isolated from adult hematopoietic organs as well on developmental early precursors of these cells. Its expression increases with more primitive phenotype of cells and it may explain that our group noticed its high expression in addition to HSC and EPC on human very small CD133+CD34+Lin-CD45– cells, which can be specified as reported by us and others into functional HSCs and EPCs. COVID19 after binding to ACE2 may hyperactivate Nlrp3 inflammasome as we recently demonstrated in cells at different level of specification into hematopoietic and endothelial lineage. This may lead to pyroptotic death of the cells exposed to virus SP. Moreover, this could lead also as we postulated of an initiation of “cytokine storm” by innate immunity cells. Evidence accumulates that COVID19 infection despite a fact that it manifests primarily as a respiratory syndrome has significant impact on other organs including the hematopoietic system and endothelium leading to several complications. To support this a large percentage of infected patients, suffer from lymphopenia and thrombocytopenia as well as from damage of endothelium that promotes hypercoagulability. Nevertheless, there are still not very well-known mechanisms how virus affect human stem cells and damage them by productive or abortive infection. It is well known that the innate immune response and activation of the Nlrp3 inflammasome are important defense mechanisms during the first days of infection, until acquired immunity responds with the production of antibodies. However, as mentioned above hyperactivation of this intracellular protein complex in innate cells may induce a cytokine storm or may lead to their death of other cells in mechanism of pyroptosis. Virus may also damage cells by lysis or theoretically what we hypothesize may stay after entry into long living stem cells in a latent form and become activated when immune system becomes impaired. Our group postulated a possibility that damage of stem cells for hemato/endothelial lineage may occur mainly by hyperactivation of Nlrp3 inflammasome after binding of viral SP to ACE2 expressed on these cells. Similar role may play interaction of SP with Toll like receptor-4 (TLR4). Our group and group of Dr. Hal Broxmeyer has demonstrated that exposure of umbilical cord blood-derived HSCs to SP protein decreases viability and in vitro clonogenicity of these cells. We also observed similar effect on proliferation of human EPC. Based on this a central hypothesis of our proposal is that COVID19 infection may damage by SP-ACE2 or SP-TLR4 interaction stem cells from hematopoietic/endothelial lineage which contributes to early and late consequences of this infection. Project was supported by the Polish National Center OPUS grant UM-2021/41/BNZ6/01590 RNAseq database  Patient COVID - PRJNA1167900 - <a href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1167900">https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1167900</a> scRNA-seq of VSELs: PRJNA1126429 (<a title="https://www.ncbi.nlm.nih.gov/sra/PRJNA1126429" href="https://www.ncbi.nlm.nih.gov/sra/PRJNA1126429" target="_blank" rel="noopener">https://www.ncbi.nlm.nih.gov/sra/PRJNA1126429</a>) scRNA-seq of HSC: PRJNA1128409 (<a title="https://www.ncbi.nlm.nih.gov/sra/PRJNA1128409" href="https://www.ncbi.nlm.nih.gov/sra/PRJNA1128409" target="_blank" rel="noopener">https://www.ncbi.nlm.nih.gov/sra/PRJNA1128409</a>).

F I G U R E 4 in Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus

F I G U R E 4 Double Immunofluorescence of brdu plus ki67 in the retina of Misgurnus anguillicaudatus (): (a), (b) brdu labelled cells at 7 months and (c), (d) ki67 labelled cells at 7 months; (e), (f) brdu labelled cells at 1 year and (g), (h) ki67 labelled cells at 1 year. The pigment epithelium showed positive reaction at both ages as well as a few positively reacted cells at the ganglion cell layer at the age of 7 months only. PE, Pigment epithelium layer; g, ganglion cell layer; vl, photoreceptor layer. The total number of fish used was n = 10

F I G U R E 3 in Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus

F I G U R E 3 Double immunofluorescence of brdu plus ki67 in the retina of Misgurnus anguillicaudatus at age of 3 months (). The pigment epithelium layer and the ganglion cell layer showed some proliferative cells. PE, Pigment epithelium layer; vl, photoreceptor layer; IN, inner nuclear layer; ip, inner plexiform layer; ON, outer nuclear layer; OP, outer plexiform layer; g, ganglion cell layer. The total number of fish used was n = 5

F I G U R E 5 in Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus

F I G U R E 5 Immunofluorescence for glial fibrillary acidic protein (GFAP) at the age of 1 month in the eye of Misgurnus anguillicaudatus: (a) glial cells in the periphery of lens (), (b) ganglion cell layer (), (c) inner and outer plexiform layers, photoreceptor layer and pigment epithelium layer (). Immunofluorescence for proliferating cell nuclear antigen (PCNA) in (d) glial cells in the periphery of lens (), (e) ganglion cell layer (), (f) inner and outer plexiform layers, photoreceptor layer and pigment epithelium layer (). L, Lens; PE, pigment epithelium layer; g, ganglion cell layer. The total number of fish used was n = 5

F I G U R E 2 in Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus

F I G U R E 2 The localisation in the retina of Misgurnus anguillicaudatus of (a), (d), (g), (j) bmi1, (b), (e), (h), (k) msi1 and (c), (f), (i), (l) sox2 genes () by using fluorescent in situ hybridisation: (a), (b), (c) 1 month old; (d), (e), (f) age of 7 months; (g), (h), (i) age of 1 year. The visual layer and some few pigment epithelial cells of retina, as well as the lens were the obvious sites for genes expressions. (j), (k), (l) Negative control sense probe. L, Lens; g, ganglion cell layer; vl, visual layer or photoreceptor layer; PE, pigment epithelium. The total number of fish used was n = 15

F I G U R E 1 in Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus

F I G U R E 1 General histological observations of the eye of Misgurnus anguillicaudatus, showing (a) the lens (L) and retina (R) at age 1 month stained with haematoxylin and eosin (H&E); (b) the seven layers of retina at age 1 month stained with H&E (1, ganglion cell layer; 2, inner plexiform layer; 3, inner nuclear layer; 4, outer plexiform layer; 5, outer nuclear layer; 6, photoreceptor layer or visual laye; 7, pigment epithelium); (c) retina at age 1 month stained with Holzer's crystal violet stain (, glial cells); (d) at age 1 year stained with Holzer's crystal violet stain

F I G U R E 6 in Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus

F I G U R E 6 The immunohistochemistry of (a), (b) peroxisome proliferator-activated receptor (PPAR)α and (c), (d) PPARγ in the eye () of Misgurnus anguillicaudatus. The main sites for PPARα expression were ganglion cell layer, inner plexiform, outer plexiform and visual layers. R, Retina; C, cornea; L, lens (L); GCL, ganglion cell layer; IP, inner plexiform IN, inner nuclear; OP, outer plexiform; ON, outer nuclear; vl, photoreceptor layer; PE, pigment epithelium layer (PE). The total number of fish used was n = 5

opencc-by-4.0Aug 2024View details →

Human umbilical cord mesenchymal stem cell-derived exosomes combined with mouse nerve growth factor can more effectively ameliorate the motor disorder and brain pathological injury in mice with cerebral palsy

Supplementary Table 1.The statistics of the  pole-climbing experiments and the horizontal network test Supplementary Table 2.Results of Kruskal–Wallis test as presented in pole-climbing experiments and the horizontal network test. Supplementary Figure 1. Identification of human umbilical cord mesenchymal stem cell exosomes (hUC-MSCs-exos) Supplementary Figure 2. Results of horizontal network test and pole-climbing experiments.

opencc-by-4.0Aug 2024View details →

Human umbilical cord mesenchymal stem cell-derived exosomes combined with mouse nerve growth factor can more effectively ameliorate themotor disorder and brain pathological injury inmice with cerebral palsy

This research design.

opencc-zeroOct 2021View details →

Ascorbic acid supports ex vivo generation of plasmacytoid dendritic cells from circulating hematopoietic stem cells: RNA-seq dataset

Plasmacytoid dendritic cells (pDCs) constitute a rare type of immune cell with multifaceted functions, but their potential use as a cell-based immunotherapy is challenged by the scarce cell numbers that can be extracted from blood. Here, we systematically investigate culture parameters for generating pDCs from hematopoietic stem and progenitor cells (HSPCs). Using optimized conditions combined with implementation of HSPC pre-expansion, we generate an average of 465 million HSPC-derived pDCs (HSPC-pDCs) starting from 100,000 cord blood-derived HSPCs. Furthermore, we demonstrate that such protocol allows HSPC-pDC generation from whole blood HSPCs, and these cells display a pDC phenotype and function. Using GMP compliant medium, we observe a remarkable loss of TLR7/9 responses, which is rescued by ascorbic acid supplementation. Ascorbic acid induces transcriptional signatures associated with pDC-specific innate immune pathways suggesting an undescribed role of ascorbic acid for pDC functionality. This constitutes the first protocol for generating pDCs from whole blood, and lay the foundation for investigating HSPC-pDCs for cell-based immunotherapy.