Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A millifluidic bioreactor allows the long term culture of primary lymphocytes or CD34 + hematopoietic cells while allowing the detection of tumorigenic expansion

doi: 10.3389/fbioe.2024.1388312

Figure Lengend Snippet: Prospective model for in vitro safety assessment of gene-edited human hematopoietic stem and progenitor cells (hHSPCs). The MOAB system aims to be used to test the expansion of cells bearing an unwanted protumorigenic mutation in gene-editing procedures. In case the test does not detect a significative expansion of cells, the autologous transplantation can be performed (top right), conversely if the test highlights a malignant phenotype, the cells cannot be employed for clinical applications and the autologous transplantation cannot be performed (Bottom, right). Created in BioRender.com .

Article Snippet: CD34 + HSPCs were isolated through magnetic cell separation using the CD34 MicroBead Kit UltraPure (Miltenyi Biotec, Italy), MS Columns (Miltenyi Biotec, Italy), and a MACS Separator (Miltenyi Biotec, Italy).

Techniques: In Vitro, Mutagenesis, Transplantation Assay

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Rapid process to differentiate iPSCs into iEPCs. ( A ) Schematic representation of the protocol for the differentiation of hiPSCs to iEPCs. ( B ) The cell morphology from day 1 to day 4 during differentiation. Scale bar, 50 μm. ( C ) The cells were treated with different concentrations of VEGF (0 ng/ml, 25 ng/ml, 50 ng/ml, or 100 ng/ml) from days 2–3. Representative FACS plots for iPSC-derived cells on day 4 are shown. ( D ) Changes in the gene expression of OCT4, NANOG, T, SCL, KDR, PECAM1, CD34 and CDH5 during differentiation. The results are shown as the mean ± SD of 3 independent experiments. ( E ) Immunofluorescence staining for CD31, CD34, and VE-CADHERIN on day 4. Nuclei were counterstained with DAPI. Scale bar, 50 μm. *** p < 0.001 and **** p < 0.0001.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Derivative Assay, Gene Expression, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Generation of iLSECs from iEPCs. ( A ) Schematic of the differentiation from hiPSC-derived EPCs into LSECs. ( B ) Expression levels of LSEC gene markers in CD34 sorted and no-sorted cells at D8. The results are shown as the mean ± SD of 3 independent experiments. **p < 0.01, ***p < 0.001. ( C ) FACS analysis of differentiated LSECs. Representative plots are shown. ( D ) Gene expression of STAB2, LYVE1, FCGR2B, and CD36 in hiPSC-derived LSEC-like cells cultured in differentiation medium A, B and C under hypoxic (5% O2) or normoxic conditions (21% O2). The results are shown as the mean ± SD of 3 independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns not significant. ( E ) Percentages of LYVE1 + cells at D4 and D8 (n = 5). The results are shown as representative plots for purified D8 cells. On D4, 0.92 ± 0.57% of the cells were LYVE1 + cells, and on D8, 76.94 ± 5.16% were LYVE1 + cells.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Derivative Assay, Expressing, Gene Expression, Cell Culture, Purification

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Evaluation of iLSECs. ( A ) The morphology of iLSEC-like cells after CD34 sorting. Scale bar, 100 μm. ( B ) qPCR analysis of canonical LSEC gene markers in iLSEC-like cells and HUVECs. The results are shown as the mean ± SD of 3 independent experiments. ** p < 0.01, *** p < 0.001 and **** p < 0.0001. ( C ) qRT‒PCR analysis of novel LSEC-specific marker genes (screened by ScRNA-seq analyses) in iLSECs and HUVECs. The results are shown as the mean ± SD of 3 independent experiments. * p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001. ( D ) Immunostaining of LYVE-1 (green) in the iLSECs revealed irregular round or oval-shaped perforations. The nuclei were counterstained with DAPI. Scale bar, 50 μm. E Fluorescence microscopy analysis of Dil-AcLDL and CyTM5-conjugated IgG uptake. hiPSCs, purified iLSEC-like cells and HUVECs were cultured in medium supplemented with DiI-ac-LDL or CyTM5-conjugated IgG. Scale bar, 100 μm.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Marker, Immunostaining, Fluorescence, Microscopy, Purification, Cell Culture

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Stem cell transcriptome of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst MSC, hESC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Dendrogram showing hierarchical clustering of MSC, hESC and MSC-hiPSC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing MSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC (d) and MSC-hiPSC (e) is reported in the indicated tables [Fisher's exact test].

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Mesenchymal potential of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst different F-hiPSC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Principal Component Analysis (PCA) showing 3D visualization of Principal Component (PC) 1, PC2 and PC3 of differentially expressed genes for different F-hiPSC, MSC-hiPSC and hESC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing F-hiPSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC-hiPSC (d) and F-hiPSC (e) is reported in the indicated tables [Fisher's exact test]. f) Schematic of the differentiation protocol toward MSC-like cells. g) Representative images of adipogenic (A, scale bar is 50 µm), osteogenic (O, scale bar is 50 µm) and chondrogenic (C, scale bar is 400 µm) mesenchymal derivatives. h) Left panel: representative density plot showing CD45 + hematopoietic cells (P3) gated from total cells of the cobblestone area-forming cell assay; FSC-A, forward scatter area, a.u., arbitrary units. Right panel: representative histograms showing CD34 + hematopoietic progenitor subpopulation (purple) of CD45 + cells compared to unstained control (grey); the vertical axis represents event percentage count (Count%).

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test, Control

Journal: Journal of medical genetics

Article Title: Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms.

doi: 10.1136/jmedgenet-2011-100674

Figure Lengend Snippet: Figure 1 (A) Pedigree and mutational analysis of index patient UPN 5869. The pedigree indicates a hereditary breast and ovarian cancer syndrome. Electropherograms illustrate the BRCA1 c.3112G/T mutation (arrow) as well as a nearby single nucleotide polymorphism (SNP) in DNA from fibroblasts and CD34+ leukaemic cells. Both mutation and SNP became homozygous in the leukaemic clone. The SNP array reveals a copy number state 1 and loss of heterozygosity (LOH) at the BRCA1 locus on chromosome 17q of CD34+ leukaemic blasts. (B) Pedigree and mutational analysis of index patient UPN 6371. The pedigree is not specific for a certain syndrome. Electropherograms illustrate the TP53 c.845_848dupGGCG mutation in germline as well as somatic DNA. SNP array shows copy number state 1 and LOH at the TP53 locus on chromosome 17p of CD34+ leukaemic cells. Filled symbols, subjects with malignancies; open symbols, asymptomatic subjects; the arrow indicates the index patient; number within symbol corresponds to the number of asymptomatic individuals. Red regions represent losses and violet regions LOH. BlaCa, bladder cancer; BrCa, breast cancer; Ca, cancer of unknown origin; GaCa, gastric cancer; Leuk, leukaemia of unknown type; Pheo, pheochromocytoma; Plas, plasmacytoma; ProCa, prostate cancer; UtCa, uterine cancer; y, age in years at diagnosis.

Article Snippet: CD34+ cells were separated from diagnostic blood or bone marrow specimens using the magnetic activated cell sorting CD34 MicroBead Kit together with MS Columns and MiniMACS Separator (all Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Mutagenesis, Biomarker Discovery

Journal: Stem Cells International

Article Title: Proliferin-1 Ameliorates Cardiotoxin-Related Skeletal Muscle Repair in Mice

doi: 10.1155/2021/9202990

Figure Lengend Snippet: rPLF-1 alleviated the expressions of desmin and laminin proteins in the gastrocnemius muscles at the indicated days after CTX injection. (a, b) Double immunofluorescence was performed with the mouse mAb against desmin ( red ) and the rabbit polyclonal antibody (pAb) against laminin-5 ( green ). Representative images and quantitative data of the desmin protein expression in the gastrocnemius muscle of both experimental groups. (c) Double immunofluorescence was performed with goat pAb against integrin- α 7 ( red ) and rabbit mAb against CD34 ( green ). Representative images and quantitative data show the numbers of CD34 + /integrin- α 7 + muscle stem cells (MuSCs). (d) Immunostaining was performed with mouse mAb against mouse monoclonal proliferating cell nuclear antigen (PCNA). Representative images and quantitative data show PCNA-positive cells. Red arrowheads : CD34 + /integrin- α 7 + cells or PCNA + cells. Data are mean ± SE ( n = 6–7). ∗ p < 0.05 and ∗∗ p < 0.01 vs. controls by Student's t -test.

Article Snippet: The fluorescein isothiocyanate- (FITC-) labeled CD34 antibody was purchased from eBioscience (San Diego, CA).

Techniques: Muscles, Injection, Immunofluorescence, Expressing, Immunostaining

Journal: Stem Cells International

Article Title: Proliferin-1 Ameliorates Cardiotoxin-Related Skeletal Muscle Repair in Mice

doi: 10.1155/2021/9202990

Figure Lengend Snippet: rPLF-1 stimulated bone marrow (BM) MuSC production and mobilization in response to CTX injury. (a, b) Representative dot plots and quantitative data for the numbers of CD34 + /integrin- α 7 + MuSCs in BM and peripheral blood (PB). (c) After the isolation of BM-derived integrin- α 7 + stem cells with magnetic beads, the cells were cultured on cover glasses for 24 hr and then subjected to double immunofluorescence with goat pAb against integrin- α 7 ( red ) and mouse mAb against Ki67 ( green ). Representative double fluorescence images and quantitative data show the numbers of proliferating cells (×200 magnification). Results are mean ± SE ( n = 7–8). ∗∗ p < 0.01 vs. corresponding control groups by Student's t -test.

Article Snippet: The fluorescein isothiocyanate- (FITC-) labeled CD34 antibody was purchased from eBioscience (San Diego, CA).

Techniques: Isolation, Derivative Assay, Magnetic Beads, Cell Culture, Immunofluorescence, Fluorescence, Control

Journal: Stem Cells International

Article Title: Proliferin-1 Ameliorates Cardiotoxin-Related Skeletal Muscle Repair in Mice

doi: 10.1155/2021/9202990

Figure Lengend Snippet: PLF-1 blocking accelerated the impaired desmin and laminin expressions in the gastrocnemius muscles postinjury. (a, b) Double immunofluorescence was performed with the mouse monoclonal antibody (mAb) against desmin ( red ) and the rabbit polyclonal antibody (pAb) against laminin-5 ( green ). Representative images and quantitative data show the contents of desmin proteins in the gastrocnemius of both experimental groups. (c) Double immunofluorescence was performed with goat pAb against integrin- α 7 ( red ) and rabbit mAb against CD34 ( green ). Representative images and quantitative data show the numbers of CD34 + /integrin- α 7 + muscle stem cells (MuSCs) in the gastrocnemius muscles of both experimental groups. (d) Immunostaining was performed with mouse mAb against proliferating cell nuclear antigen (PCNA). Representative images and quantitative data show PCNA-positive cells in the gastrocnemius muscles of both groups. Red arrowheads : CD34 + /integrin- α 7 + cells or PCNA + . Data are mean ± SE ( n = 7–8). ∗ p < 0.05 and ∗∗ p < 0.01 vs. controls by Student's t -test.

Article Snippet: The fluorescein isothiocyanate- (FITC-) labeled CD34 antibody was purchased from eBioscience (San Diego, CA).

Techniques: Blocking Assay, Muscles, Immunofluorescence, Immunostaining

Journal: Stem Cells International

Article Title: Proliferin-1 Ameliorates Cardiotoxin-Related Skeletal Muscle Repair in Mice

doi: 10.1155/2021/9202990

Figure Lengend Snippet: PLF-1 depletion reduced the numbers of MuSCs in bone marrow (BM) and peripheral blood (PB) in response to CTX injury. (a, b) Representative dot plots and quantitative data for the numbers of CD34 + /integrin- α 7 + MuSCs in BM and PB of both experimental groups. (c) After the isolation of BM-derived integrin- α 7 + stem cells with magnetic beads, the cells were cultured on cover glasses for 24 hr and then subjected to double immunofluorescence with goat pAb against integrin- α 7 ( red ) and mouse mAb against Ki67 ( green ). Representative double images and quantitative data show the numbers of proliferating cells (×200 magnification). Results are mean ± SE ( n = 6–8). ∗∗ p < 0.01 vs. corresponding control groups by Student's t -test.

Article Snippet: The fluorescein isothiocyanate- (FITC-) labeled CD34 antibody was purchased from eBioscience (San Diego, CA).

Techniques: Isolation, Derivative Assay, Magnetic Beads, Cell Culture, Immunofluorescence, Control

Journal: Cancers

Article Title: Increased Plasma Levels of lncRNAs LINC01268 , GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis

doi: 10.3390/cancers13194744

Figure Lengend Snippet: PCA and Hierarchical clustering of OpenArray Data. ( a ) PCA graph represents the global lncRNAs expression in CD34+ stem/progenitor cells. ( b ) Hierarchical clustering of samples according to the expression of 28 lncRNA transcripts, corresponding to 26 lncRNAs. Both analyses were performed by Partek GS, version 6.6. FDR = False Discovery Rate; BM = control samples isolated from bone marrow; PB = control samples isolated from peripheral blood; HD = healthy donor; MF = myelofibrosis patients; PMF = primary myelofibrosis; SMF = secondary myelofibrosis.

Article Snippet: Human CD34+ cells were purified by using magnetic beads separation system (CD34 MicroBead Kit UltraPure, Miltenyi Biotech, Auburn, CA, USA) from peripheral blood (PB) of 83 patients (MFs).

Techniques: Expressing, Control, Isolation

Journal: Cancers

Article Title: Increased Plasma Levels of lncRNAs LINC01268 , GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis

doi: 10.3390/cancers13194744

Figure Lengend Snippet: Clinical and molecular features of MF patients included in our dataset, grouped according to the levels of single circulating lncRNAs. Data in the table are reported as n (%). p = p -value. N = evaluable samples. LDH = lactate dehydrogenase; DIPSS = Dynamic International Prognostic Score System. Significant p -value ( p < 0.05) are represented in bold. “—” = missing value.

Article Snippet: Human CD34+ cells were purified by using magnetic beads separation system (CD34 MicroBead Kit UltraPure, Miltenyi Biotech, Auburn, CA, USA) from peripheral blood (PB) of 83 patients (MFs).

Techniques: Mutagenesis, Transformation Assay

Journal: Cancers

Article Title: Increased Plasma Levels of lncRNAs LINC01268 , GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis

doi: 10.3390/cancers13194744

Figure Lengend Snippet: Correlation analysis of level of circulating lncRNAs with WBC count, circulating CD34+ count and LDH activity in MF patients. Box plots represent values from white blood cell count (WBC) ( a , d , g , j , m ), CD34+ cells count ( b , e , h , k , n ) and LDH activity ( c , f , i , l , o ) in MF samples presenting low or high levels of target RNA. The box extends from the 25th to the 75th percentiles. The line in middle of the box is plotted as the median. The whiskers are drawn as 5th and 95th percentiles. Values above or below the whiskers are plotted as individual points. Samples with low or high levels of target lncRNA are represented by blue and red boxes, respectively. p = p -value was computed by non-parametric Mann–Whitney U test.

Article Snippet: Human CD34+ cells were purified by using magnetic beads separation system (CD34 MicroBead Kit UltraPure, Miltenyi Biotech, Auburn, CA, USA) from peripheral blood (PB) of 83 patients (MFs).

Techniques: Activity Assay, Cell Counting, MANN-WHITNEY

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Impact of aging on CD146 + mesenchymal stromal cells-mediated regulation of bone marrow CD34 + hematopoietic stem/progenitor cell mobilization

doi: 10.3389/fbioe.2026.1802093

Figure Lengend Snippet: In vitro testing of the impact of age on human BMSC-mediated CD34 + HSPCs migration. (A) Schematic representation of the experimental setup, including (i) a timeline of the principal experimental steps, (ii) a graphic description of the different experimental conditions and (iii) a representative image showing migrated CD34 + HSPCs (red) in the different experimental conditions. (B) Age-dependent effects of mobilizing agents on CD146 + BMSC-mediated CD34 + HSPC transmigration; each graph represents an individual donor with its technical replicates, express as percentage of migrated cells relative to the internal reference (without CD146 + BMSCs). N = 3 subjects per group, with 3 technical replicates per subject. Statistical analysis was performed using the Kruskal–Wallis test. * p < 0.05; ** p < 0.01. Negative control, NC; hematopoietic stem progenitor cells, HSPCs; bone marrow stromal cells, BMSCs; untreated control, CTRL; specific CXCR4 inhibitor, AMD3100; granulocyte colony-stimulating factor, G-CSF; norepinephrine, NE. (Panel A (ii) and (iii) created in https://BioRender.com ).

Article Snippet: BM CD34 + HSPCs were isolated from one subject (female, 64 years old) using the Diamond CD34 + Magnetic Bead Isolation Kit (Miltenyi Biotec, cat # 130-094–531) according to the manufacturer’s instructions.

Techniques: In Vitro, Migration, Transmigration Assay, Negative Control, Control