human osteoblasts  (PromoCell)

Journal: Calcified Tissue International

Article Title: Bone from Healthy Individuals and Patients with CKD Expresses the Sodium-Glucose Co-transporter-2 (SGLT2)

doi: 10.1007/s00223-026-01498-7

Figure Lengend Snippet: Preliminary qPCR amplification for primer selection. A and B Unsuccessful preliminary qPCR amplification using 267 and 430 bp primer pairs for SLC5A2 for MG-63 cells, respectively. The amplification plot displays the performance designed to amplify as a product of the human SLC5A2 transcript ( NM_003041.4 ), with its specific binding sites shown below. While the primers yielded a clear amplification signal in the positive control HK-2 renal cells, no detectable expression was observed in the osteoblast-like MG-63 cell line. Due to the lack of amplification in MG-63 cells, these primer sets were deemed unsuitable for the study. ( C ) Successful validation of the selected 108 bp primer for SLC5A2. This primer pair shows robust and comparable amplification in both MG-63 and HK-2 cell lines between 22 and 24 cycles, confirming its suitability for subsequent comparative gene expression analysis by qPCR

Article Snippet: Human osteoblast-like (MG-63) and human kidney (HK-2) cell lines were obtained from the American Type Culture Collection (ATCC CRL-1427 and -2190, USA).

Techniques: Amplification, Selection, Binding Assay, Positive Control, Expressing, Biomarker Discovery, Gene Expression

Journal: Calcified Tissue International

Article Title: Bone from Healthy Individuals and Patients with CKD Expresses the Sodium-Glucose Co-transporter-2 (SGLT2)

doi: 10.1007/s00223-026-01498-7

Figure Lengend Snippet: Relative quantification of SLC5A2 gene expression. A Raw Ct values of two biologicals (in duplicates) showing comparable SLC5A2 expression in MG-63 and HK-2 cells; for the first time, the SLC5A2 expression in osteoblast-like MG-63 cells is confirmed; B Raw Ct values of three healthy individuals bone samples and ten bone samples from patients with CKD (in duplicates) indicating that the SLC5A2 expression is lower in bone samples from patients with CKD. After ΔΔCt normalization by GAPDH, no significant difference is seen between C cell lines (two biologicals in duplicates), or between D bone samples (mean of duplicates) from healthy subjects and patients with CKD. GAPDH variation in expression (above 3.5 Cts) prevented a suitable normalization of SLC5A2 expression in healthy subjects versus patients with CKD; thus, each condition was normalized to its own GAPDH expression for representation purposes

Article Snippet: Human osteoblast-like (MG-63) and human kidney (HK-2) cell lines were obtained from the American Type Culture Collection (ATCC CRL-1427 and -2190, USA).

Techniques: Quantitative Proteomics, Gene Expression, Expressing

Journal: Calcified Tissue International

Article Title: Bone from Healthy Individuals and Patients with CKD Expresses the Sodium-Glucose Co-transporter-2 (SGLT2)

doi: 10.1007/s00223-026-01498-7

Figure Lengend Snippet: Absolute quantification of SLC5A2 and GAPDH transcript copy numbers in cells (two biologicals in duplicates) and human bone (mean of duplicates from three healthy individuals’ bone and ten samples from patients with CKD) after 40 cycles. The use of standard curves and linear regressions using dilutions in log 10 -3, -4, -5, and -6 from experimental controls (HK-2 cells and apparently healthy individuals´ bone samples) templates allowed the estimation of an unknown number of copies for each primer individually. This analysis confirms comparable absolute SLC5A2 transcript levels between HK-2 and osteoblast-like MG-63 cells, while GAPDH levels were modestly lower in MG-63 cells. In human bone tissue, a non-significant trend towards higher SLC5A2 copies was observed in samples from patients with chronic kidney disease (CKD) compared to samples from healthy subjects. Critically, GAPDH copy numbers were significantly reduced in CKD bone, demonstrating its instability as a reference gene in this pathology and reinforcing the value of absolute quantification for accurate expression analysis while analyzing bone samples. Additionally, internal controls also using pools from biologicals of HK-2 cells and healthy bone (red dashed line indicated within each ‘y’ axis) were adopted as an extra sample (in duplicates) to corroborate the unknown individual sample results; this reference obtained its number of copies within the range of their tested biologicals

Article Snippet: Human osteoblast-like (MG-63) and human kidney (HK-2) cell lines were obtained from the American Type Culture Collection (ATCC CRL-1427 and -2190, USA).

Techniques: Quantitative Proteomics, Expressing

Journal: Calcified Tissue International

Article Title: Bone from Healthy Individuals and Patients with CKD Expresses the Sodium-Glucose Co-transporter-2 (SGLT2)

doi: 10.1007/s00223-026-01498-7

Figure Lengend Snippet: Western blot analysis of the expression of SGLT2 in four distinct biological replicates of MG-63 osteoblast-like cells. A specific band corresponding to SGLT2 was successfully detected at its predicted molecular weight of approximately 73 kDa in all four samples. Housekeeping protein GAPDH bands are uniformly observed at the expected size of 37 kDa

Article Snippet: Human osteoblast-like (MG-63) and human kidney (HK-2) cell lines were obtained from the American Type Culture Collection (ATCC CRL-1427 and -2190, USA).

Techniques: Western Blot, Expressing, Molecular Weight

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Schematic illustration of the protein-based platform for the direct osteogenic reprogramming of human dermal fibroblasts (HDFs). (A) Production of recombinant Oct4-30Kc19 and Cbfβ-30Kc19 fusion proteins. Expression plasmids encoding reprogramming factors fused with the 30Kc19 moiety were introduced into an E. coli expression system to generate cell-permeable recombinant proteins. (B) Direct osteogenic reprogramming via intracellular protein transduction. The fusion proteins are intracellularly delivered through 30Kc19-mediated transport. Oct4-30Kc19 induces a state of cellular plasticity, while Cbfβ-30Kc19 promotes osteogenic lineage commitment, synergistically driving the conversion of HDFs into functional osteoblasts.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Recombinant, Expressing, Transduction, Functional Assay

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Direct reprogramming of HDFs into osteoblasts through ectopic overexpression of Oct4 and Cbfβ. (A) Schematic illustration of direct reprogramming process using the pCXLE episomal plasmid delivery system. The pCXLE-Oct4 and pCXLE-Cbfβ plasmids were delivered into HDFs via cationic polymer-based transfection, either individually or in combination. (B) Representative images of alkaline phosphatase (ALP) staining after 14 d of culture in osteogenic medium (OM). (C and D) Calcium deposition after 24 d of osteogenic induction, as detected by Alizarin Red S (ARS) and OsteoImage assays. (E and F) Immunofluorescence images showing expression of osteopontin (OPN) and osteocalcin (OCN), on day 24. Coexpression of Oct4 and Cbfβ induced robust expression of both osteogenic markers.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Over Expression, Plasmid Preparation, Polymer, Transfection, Staining, Immunofluorescence, Expressing

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Direct reprogramming of HDFs into osteoblasts using cell-permeable protein-based platform. (A) Schematic illustration of the reprogramming strategy using 30Kc19-fused recombinant proteins. HDFs were treated with a combination of Oct4-30Kc19 and Cbfβ-30Kc19 recombinant proteins 8 times over 8 d, followed by culture in OM. (B and C) ARS staining and subsequent quantification on day 24, showing robust calcium deposition in the group treated with both proteins. Data are presented as means ± SD ( n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. Unless otherwise indicated, comparisons were made to the nontreated control group (** P < 0.01; *** P < 0.001; ns, not significant). (D and E) Von Kossa staining and OsteoImage mineralization assay images, confirming mineralized matrix formation. Treatment with both Oct4-30Kc19 and Cbfβ-30Kc19 resulted in significantly enhanced mineralization compared with single-factor or untreated controls.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Recombinant, Staining, Control, Mineralization Assay

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Transcriptomic remodeling toward an osteoblast lineage by Oct4-30Kc19 and Cbfβ-30Kc19 fusion proteins. (A) Scatterplot showing differentially expressed genes (DEGs) between nontreated HDFs and protein-induced osteoblasts (piOBs) treated with Oct4-30Kc19 and Cbfβ-30Kc19 proteins (fold change ≥ 2). (B) Gene Ontology (GO) enrichment analysis of DEGs, highlighting overrepresentation of transcription-related biological processes. (C) Protein interaction network of enriched GO terms related to transcription regulation by RNA polymerase II. (D) Hierarchical clustering analysis of genes differentially expressed in both piOBs and primary human osteoblasts (hOBs), relative to HDFs. (E) Heatmap of DEGs associated with the ossification GO term, showing the expression of key osteogenic markers.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Biomaterials Research

Article Title: Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

doi: 10.34133/bmr.0320

Figure Lengend Snippet: Efficient bone defect regeneration using a cell-permeable protein-based direct reprogramming platform. (A) Schematic illustration of the in vivo bone regeneration experiment. HDFs were pretreated 8 times with Oct4-30Kc19 and Cbfβ-30Kc19 proteins, seeded onto gelatin cryogels, and transplanted into 4-mm-sized calvarial defects in mice. Created with biorender.com . (B) Representative micro-CT 3D images showing bone regeneration 8 weeks post-transplantation. Green areas and arrows indicate newly regenerated bones, while yellow arrows denote bone defect regions. (C and D) Quantification of bone volume fraction (BV/TV) and trabecular separation in the regenerated bone tissue. Data are presented as means ± SD ( n = 4). Statistical significance was determined by Student’s t test (*** P < 0.001). (E) Hematoxylin and eosin (H&E) staining showing histological differences between defects implanted with untreated HDFs and those implanted with piOBs. (F) Masson’s trichrome (MTC) staining revealing collagen-rich new bone formation in the piOB-treated group. FT, fibrous tissues; NB, new bones. (G and H) Immunofluorescent staining for OPN and OCN, confirming the presence of mature osteoblast-derived matrix in piOB-transplanted defects.

Article Snippet: Primary human osteoblasts (hOBs; PromoCell, Heidelberg, Germany) were cultured in Osteoblast Growth Medium (PromoCell) according to the manufacturer’s instructions.

Techniques: In Vivo, Micro-CT, Transplantation Assay, Staining, Derivative Assay