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Validation of XYLT1 gene editing <t>in</t> <t>hMSCs.</t> ( A ) Sanger sequencing analysis confirmed the successful disruption of the XYLT1 gene (Gene ID: 64131) at the targeted Cas9 cleavage site within exon 3. The electropherogram reveals overlapping signals, indicative of a mixed population of DNA sequences. This suggests the presence of both edited and unedited alleles. ( B ) The T7EI assay demonstrates the presence of CRISPR-Cas9-induced mutations. The DNA fragments amplified from the targeted XYLT1 locus were subjected to heteroduplex formation and cleavage by T7EI. Negative controls without DNA were included for both the PCR amplification (NC PCR) and T7 endonuclease assay (NC T7). By analyzing the cleaved DNA fragments via gel electrophoresis of the samples derived from RNP complex-treated cells, distinct cleavage bands are shown to be present, indicating successful gene editing, whereas the untreated control displays a single DNA fragment. The presence and intensity of the cleaved bands indicate the frequency of indel formation at the target site. Mutation rates were quantified based on band intensities using ImageJ software. M = marker, WT = WT <t>hMSC</t> culture, KD = KD hMSC culture (#1, #2). ( C ) TA cloning of the PCR products, followed by sequencing, provided further validation of indels at the XYLT1 target site. The XYLT1 gRNA sequence (blue) for complementary binding in exon 3 and the PAM sequence (red) required for Cas9 binding are highlighted. Representative clones (left side) exhibit a range of mutation types, including small insertions and deletions, underscoring the efficiency and diversity of CRISPR-Cas9-mediated gene disruption. These indels can induce a frameshift, leading to a premature stop codon and resulting in a truncated amino acid sequence (AS) of the protein (right side).

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1) Product Images from "XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation"

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms26157363

Validation of XYLT1 gene editing in hMSCs. ( A ) Sanger sequencing analysis confirmed the successful disruption of the XYLT1 gene (Gene ID: 64131) at the targeted Cas9 cleavage site within exon 3. The electropherogram reveals overlapping signals, indicative of a mixed population of DNA sequences. This suggests the presence of both edited and unedited alleles. ( B ) The T7EI assay demonstrates the presence of CRISPR-Cas9-induced mutations. The DNA fragments amplified from the targeted XYLT1 locus were subjected to heteroduplex formation and cleavage by T7EI. Negative controls without DNA were included for both the PCR amplification (NC PCR) and T7 endonuclease assay (NC T7). By analyzing the cleaved DNA fragments via gel electrophoresis of the samples derived from RNP complex-treated cells, distinct cleavage bands are shown to be present, indicating successful gene editing, whereas the untreated control displays a single DNA fragment. The presence and intensity of the cleaved bands indicate the frequency of indel formation at the target site. Mutation rates were quantified based on band intensities using ImageJ software. M = marker, WT = WT hMSC culture, KD = KD hMSC culture (#1, #2). ( C ) TA cloning of the PCR products, followed by sequencing, provided further validation of indels at the XYLT1 target site. The XYLT1 gRNA sequence (blue) for complementary binding in exon 3 and the PAM sequence (red) required for Cas9 binding are highlighted. Representative clones (left side) exhibit a range of mutation types, including small insertions and deletions, underscoring the efficiency and diversity of CRISPR-Cas9-mediated gene disruption. These indels can induce a frameshift, leading to a premature stop codon and resulting in a truncated amino acid sequence (AS) of the protein (right side).

Figure Legend Snippet: Validation of XYLT1 gene editing in hMSCs. ( A ) Sanger sequencing analysis confirmed the successful disruption of the XYLT1 gene (Gene ID: 64131) at the targeted Cas9 cleavage site within exon 3. The electropherogram reveals overlapping signals, indicative of a mixed population of DNA sequences. This suggests the presence of both edited and unedited alleles. ( B ) The T7EI assay demonstrates the presence of CRISPR-Cas9-induced mutations. The DNA fragments amplified from the targeted XYLT1 locus were subjected to heteroduplex formation and cleavage by T7EI. Negative controls without DNA were included for both the PCR amplification (NC PCR) and T7 endonuclease assay (NC T7). By analyzing the cleaved DNA fragments via gel electrophoresis of the samples derived from RNP complex-treated cells, distinct cleavage bands are shown to be present, indicating successful gene editing, whereas the untreated control displays a single DNA fragment. The presence and intensity of the cleaved bands indicate the frequency of indel formation at the target site. Mutation rates were quantified based on band intensities using ImageJ software. M = marker, WT = WT hMSC culture, KD = KD hMSC culture (#1, #2). ( C ) TA cloning of the PCR products, followed by sequencing, provided further validation of indels at the XYLT1 target site. The XYLT1 gRNA sequence (blue) for complementary binding in exon 3 and the PAM sequence (red) required for Cas9 binding are highlighted. Representative clones (left side) exhibit a range of mutation types, including small insertions and deletions, underscoring the efficiency and diversity of CRISPR-Cas9-mediated gene disruption. These indels can induce a frameshift, leading to a premature stop codon and resulting in a truncated amino acid sequence (AS) of the protein (right side).

Techniques Used: Biomarker Discovery, Sequencing, Disruption, T7EI Assay, CRISPR, Amplification, Nucleic Acid Electrophoresis, Derivative Assay, Control, Mutagenesis, Software, Marker, TA Cloning, Binding Assay, Clone Assay

Quantification of XYLT1, XYLT2 mRNA expression, and XT-I activity in chondrogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were formed into spheroids and cultured in either standard (control) or chondrogenic differentiation medium (differentiation) for 2 or 7 days. The mRNA levels of ( A ) XYLT1 and ( B ) XYLT2 were assessed at day 2 via qRT-PCR. ( C ) The XT-I enzyme activity per sample volume (nU/volume) was measured at days 2 and 7 using the mass spectrometric XT-I assay. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 (A, B) or n = 2 ( C ) technical replicates per biological replicate, normalized to the WT sample. Mann–Whitney ( A , B ) and Kruskal–Wallis ( C ) test significance levels: not significant (ns), p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****).

Figure Legend Snippet: Quantification of XYLT1, XYLT2 mRNA expression, and XT-I activity in chondrogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were formed into spheroids and cultured in either standard (control) or chondrogenic differentiation medium (differentiation) for 2 or 7 days. The mRNA levels of ( A ) XYLT1 and ( B ) XYLT2 were assessed at day 2 via qRT-PCR. ( C ) The XT-I enzyme activity per sample volume (nU/volume) was measured at days 2 and 7 using the mass spectrometric XT-I assay. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 (A, B) or n = 2 ( C ) technical replicates per biological replicate, normalized to the WT sample. Mann–Whitney ( A , B ) and Kruskal–Wallis ( C ) test significance levels: not significant (ns), p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****).

Techniques Used: Expressing, Activity Assay, Cell Culture, Transfection, Control, Quantitative RT-PCR, MANN-WHITNEY

Quantification of XYLT1 , XYLT2 mRNA expression, and XT-I activity in osteogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured post-transfection to 90% confluence, then seeded at 100 cells/mm 2 and grown to 100% confluence. Cultivation continued for 7 or 14 days in either standard (control) or osteogenic differentiation medium (differentiated). The mRNA levels of ( A ) XYLT1 and ( B ) XYLT2 were assessed via qRT-PCR, while ( C ) XT-I enzyme activity (nU/ng) was determined using the mass spectrometric XT-I assay, normalized to DNA content. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 ( A , B ) or n = 2 ( C ) technical replicates per biological replicate, normalized to the WT controls. Kruskal–Wallis test significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Figure Legend Snippet: Quantification of XYLT1 , XYLT2 mRNA expression, and XT-I activity in osteogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured post-transfection to 90% confluence, then seeded at 100 cells/mm 2 and grown to 100% confluence. Cultivation continued for 7 or 14 days in either standard (control) or osteogenic differentiation medium (differentiated). The mRNA levels of ( A ) XYLT1 and ( B ) XYLT2 were assessed via qRT-PCR, while ( C ) XT-I enzyme activity (nU/ng) was determined using the mass spectrometric XT-I assay, normalized to DNA content. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 ( A , B ) or n = 2 ( C ) technical replicates per biological replicate, normalized to the WT controls. Kruskal–Wallis test significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Techniques Used: Expressing, Activity Assay, Cell Culture, Transfection, Control, Quantitative RT-PCR

Quantification of XYLT1, XYLT2 mRNA expression, and XT-I activity in adipogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured post-transfection with (KD) or without (WT) the RNP complex until reaching 90% confluency. Cells were seeded at a density of 210 cells/mm 2 and cultured in standard hMSC medium until 100% confluency. Adipogenesis was induced over a period of twenty days by culturing the cells for three cycles of three days in the induction medium, followed by one day in the maintenance medium. Subsequently, cells were cultured in maintenance medium for an additional seven days. The mRNA expression levels of ( A , C ) XYLT1 and ( B , D ) XYLT2 were assessed at day 3 (n = 2 primary cell cultures) and day 20 (n = 1 primary cell culture) via qRT-PCR. ( E ) XT-I enzyme activity (nU/ng) was measured at day 20 (n = 1 primary cell culture) using the mass spectrometric XT-I assay, normalized to DNA content. Data are means ± SEM from n = 3 biological replicates per primary cell culture and n = 3 ( A – D ) or n = 2 ( E ) technical replicates per biological replicate, normalized to the WT sample. Mann–Whitney test significance levels: not significant (ns), (*) p < 0.05, (**) p < 0.01.

Figure Legend Snippet: Quantification of XYLT1, XYLT2 mRNA expression, and XT-I activity in adipogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured post-transfection with (KD) or without (WT) the RNP complex until reaching 90% confluency. Cells were seeded at a density of 210 cells/mm 2 and cultured in standard hMSC medium until 100% confluency. Adipogenesis was induced over a period of twenty days by culturing the cells for three cycles of three days in the induction medium, followed by one day in the maintenance medium. Subsequently, cells were cultured in maintenance medium for an additional seven days. The mRNA expression levels of ( A , C ) XYLT1 and ( B , D ) XYLT2 were assessed at day 3 (n = 2 primary cell cultures) and day 20 (n = 1 primary cell culture) via qRT-PCR. ( E ) XT-I enzyme activity (nU/ng) was measured at day 20 (n = 1 primary cell culture) using the mass spectrometric XT-I assay, normalized to DNA content. Data are means ± SEM from n = 3 biological replicates per primary cell culture and n = 3 ( A – D ) or n = 2 ( E ) technical replicates per biological replicate, normalized to the WT sample. Mann–Whitney test significance levels: not significant (ns), (*) p < 0.05, (**) p < 0.01.

Techniques Used: Expressing, Activity Assay, Cell Culture, Transfection, Quantitative RT-PCR, MANN-WHITNEY

XYLT1 deficiency alters the ECM deposition/PG content of chondrogenically differentiated hMSCs. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were formed into spheroids and cultured in either hMSC standard medium (control) or chondrogenic differentiation medium (differentiation). The pellet cultures were fixed after 14 or 28 days and incubated in a 15% sucrose solution, and 5 μm cryosections were prepared. Cryosections were stained with a 1% alcian blue solution to visualize acidic PG and glycoproteins. Cell nuclei were counterstained blue to purple using Gill’s III hematoxylin solution. Representative images are shown from n = 2 primary cell cultures (indicated with #1, #2), n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Scale bar: 250 μm.

Figure Legend Snippet: XYLT1 deficiency alters the ECM deposition/PG content of chondrogenically differentiated hMSCs. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were formed into spheroids and cultured in either hMSC standard medium (control) or chondrogenic differentiation medium (differentiation). The pellet cultures were fixed after 14 or 28 days and incubated in a 15% sucrose solution, and 5 μm cryosections were prepared. Cryosections were stained with a 1% alcian blue solution to visualize acidic PG and glycoproteins. Cell nuclei were counterstained blue to purple using Gill’s III hematoxylin solution. Representative images are shown from n = 2 primary cell cultures (indicated with #1, #2), n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Scale bar: 250 μm.

Techniques Used: Cell Culture, Transfection, Control, Incubation, Staining

XYLT1 deficiency affects the collagen deposition of chondrogenically differentiated hMSCs. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were pelleted and cultured in either hMSC standard (control) or chondrogenic differentiation (differentiation) medium. The pellet cultures were fixed after 14 or 28 days and incubated in a 15% sucrose solution, and 5 μm cryosections were prepared. Cryosections were stained with Picro-Sirius red solution to visualize collagenous structures in orange-red. Cell nuclei were stained brown-black with acid-resistant Weigert’s iron hematoxylin solution. Representative images are shown from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Scale bar: 250 μm.

Figure Legend Snippet: XYLT1 deficiency affects the collagen deposition of chondrogenically differentiated hMSCs. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were pelleted and cultured in either hMSC standard (control) or chondrogenic differentiation (differentiation) medium. The pellet cultures were fixed after 14 or 28 days and incubated in a 15% sucrose solution, and 5 μm cryosections were prepared. Cryosections were stained with Picro-Sirius red solution to visualize collagenous structures in orange-red. Cell nuclei were stained brown-black with acid-resistant Weigert’s iron hematoxylin solution. Representative images are shown from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Scale bar: 250 μm.

Techniques Used: Cell Culture, Transfection, Control, Incubation, Staining

Analysis of osteogenic differentiation in WT and KD hMSCs via qRT-PCR at days 7 and 14. Primary hMSCs were transfected either without (WT) or with (KD) the RNP complex and cultured until reaching 90% confluence. The cells were subcultured at a density of 100 cells/mm 2 , grown until 100% confluence, and then cultivated for 7 or 14 days in either hMSC standard medium (control) or osteogenic differentiation medium (differentiated). The mRNA expression levels of the ( A ) RUNX2 , ( B ) SOX9 , ( C ) OPG , ( D ) COL1A1 , ( E ) BGLAP (osteocalcin), and ( F ) SPP1 (OPN) genes were determined at both day 7 and 14 of osteogenic differentiation. Data are presented as means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate, with normalization to the respective WT control. Kruskal–Wallis significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Figure Legend Snippet: Analysis of osteogenic differentiation in WT and KD hMSCs via qRT-PCR at days 7 and 14. Primary hMSCs were transfected either without (WT) or with (KD) the RNP complex and cultured until reaching 90% confluence. The cells were subcultured at a density of 100 cells/mm 2 , grown until 100% confluence, and then cultivated for 7 or 14 days in either hMSC standard medium (control) or osteogenic differentiation medium (differentiated). The mRNA expression levels of the ( A ) RUNX2 , ( B ) SOX9 , ( C ) OPG , ( D ) COL1A1 , ( E ) BGLAP (osteocalcin), and ( F ) SPP1 (OPN) genes were determined at both day 7 and 14 of osteogenic differentiation. Data are presented as means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate, with normalization to the respective WT control. Kruskal–Wallis significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Techniques Used: Quantitative RT-PCR, Transfection, Cell Culture, Control, Expressing

Comparison of the mineralization process in WT and KD hMSCs during osteogenesis. The hMSCs were cultured post-transfection without (WT) or with (KD) RNP complex until reaching 90% confluence. Cells were subcultured at a density of 100 cells/mm 2 and, upon reaching 100% confluence, cultured for 7 or 14 days in either hMSC standard medium (control) or osteogenic differentiation medium (differentiated). ( A ) The ALP activity in cell lysates was quantified spectrophotometrically in technical duplicates. The ALP activities (mU/µg) were normalized to the protein content of cell lysates. ( B ) Free inorganic phosphate (P i ) levels (µg/ng) from cell culture supernatants were measured using the ARCHITECT c8000 clinical chemistry analyzer and normalized to the DNA content of the respective cell lysates. ( C ) Calcium deposits were visualized by Alizarin Red S staining using two biological replicates per condition and time point. Representative images of the WT and KD hMSCs cultures on day 7 are shown. Scale bar: 100 µm. ( D ) Osteopontin protein levels in conditioned media were quantified by ELISA, with differentiation controls diluted 1:10 and protein levels normalized to the respective DNA content of the cell lysates. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Kruskal–Wallis significance levels: p < 0.05 (*), p < 0.01 (**). N/A = not available.

Figure Legend Snippet: Comparison of the mineralization process in WT and KD hMSCs during osteogenesis. The hMSCs were cultured post-transfection without (WT) or with (KD) RNP complex until reaching 90% confluence. Cells were subcultured at a density of 100 cells/mm 2 and, upon reaching 100% confluence, cultured for 7 or 14 days in either hMSC standard medium (control) or osteogenic differentiation medium (differentiated). ( A ) The ALP activity in cell lysates was quantified spectrophotometrically in technical duplicates. The ALP activities (mU/µg) were normalized to the protein content of cell lysates. ( B ) Free inorganic phosphate (P i ) levels (µg/ng) from cell culture supernatants were measured using the ARCHITECT c8000 clinical chemistry analyzer and normalized to the DNA content of the respective cell lysates. ( C ) Calcium deposits were visualized by Alizarin Red S staining using two biological replicates per condition and time point. Representative images of the WT and KD hMSCs cultures on day 7 are shown. Scale bar: 100 µm. ( D ) Osteopontin protein levels in conditioned media were quantified by ELISA, with differentiation controls diluted 1:10 and protein levels normalized to the respective DNA content of the cell lysates. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Kruskal–Wallis significance levels: p < 0.05 (*), p < 0.01 (**). N/A = not available.

Techniques Used: Comparison, Cell Culture, Transfection, Control, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay

Quantification of perilipin 1 and leptin protein expression in WT and KD adipogenically differentiated hMSC cultures. The hMSCs were cultured to 90% confluence following transfection without (WT) or with (KD) the RNP complex. Cells were subcultured at a density of 210 cells/mm 2 in standard medium until reaching full confluency. Adipogenic differentiation was conducted over 20 days with cycles of induction and maintenance media, followed by 7 days in maintenance media. Controls remained in maintenance media throughout. ( A ) Cells were fixed for the fluorescence detection of perilipin 1 (magenta) with BODIPY-staining lipid droplets (yellow) and DAPI counterstaining nuclei (cyan). Scale bar = 100 μm. ( B ) Perilipin 1 expression (CTCF) was quantified using ImageJ by analyzing eight images per biological replicate, normalized to total cell number per image. ( C ) Leptin protein levels (pg/ng) in 1:10 diluted cell lysates were measured via ELISA in duplicate, normalized to total DNA. Data are means ± SEM from n = 2 primary cell cultures and n = 3 biological replicates per primary cell culture. Mann–Whitney test significance levels: not significant (ns), p < 0.05 (*).

Figure Legend Snippet: Quantification of perilipin 1 and leptin protein expression in WT and KD adipogenically differentiated hMSC cultures. The hMSCs were cultured to 90% confluence following transfection without (WT) or with (KD) the RNP complex. Cells were subcultured at a density of 210 cells/mm 2 in standard medium until reaching full confluency. Adipogenic differentiation was conducted over 20 days with cycles of induction and maintenance media, followed by 7 days in maintenance media. Controls remained in maintenance media throughout. ( A ) Cells were fixed for the fluorescence detection of perilipin 1 (magenta) with BODIPY-staining lipid droplets (yellow) and DAPI counterstaining nuclei (cyan). Scale bar = 100 μm. ( B ) Perilipin 1 expression (CTCF) was quantified using ImageJ by analyzing eight images per biological replicate, normalized to total cell number per image. ( C ) Leptin protein levels (pg/ng) in 1:10 diluted cell lysates were measured via ELISA in duplicate, normalized to total DNA. Data are means ± SEM from n = 2 primary cell cultures and n = 3 biological replicates per primary cell culture. Mann–Whitney test significance levels: not significant (ns), p < 0.05 (*).

Techniques Used: Expressing, Cell Culture, Transfection, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Image Search Results

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: Validation of XYLT1 gene editing in hMSCs. ( A ) Sanger sequencing analysis confirmed the successful disruption of the XYLT1 gene (Gene ID: 64131) at the targeted Cas9 cleavage site within exon 3. The electropherogram reveals overlapping signals, indicative of a mixed population of DNA sequences. This suggests the presence of both edited and unedited alleles. ( B ) The T7EI assay demonstrates the presence of CRISPR-Cas9-induced mutations. The DNA fragments amplified from the targeted XYLT1 locus were subjected to heteroduplex formation and cleavage by T7EI. Negative controls without DNA were included for both the PCR amplification (NC PCR) and T7 endonuclease assay (NC T7). By analyzing the cleaved DNA fragments via gel electrophoresis of the samples derived from RNP complex-treated cells, distinct cleavage bands are shown to be present, indicating successful gene editing, whereas the untreated control displays a single DNA fragment. The presence and intensity of the cleaved bands indicate the frequency of indel formation at the target site. Mutation rates were quantified based on band intensities using ImageJ software. M = marker, WT = WT hMSC culture, KD = KD hMSC culture (#1, #2). ( C ) TA cloning of the PCR products, followed by sequencing, provided further validation of indels at the XYLT1 target site. The XYLT1 gRNA sequence (blue) for complementary binding in exon 3 and the PAM sequence (red) required for Cas9 binding are highlighted. Representative clones (left side) exhibit a range of mutation types, including small insertions and deletions, underscoring the efficiency and diversity of CRISPR-Cas9-mediated gene disruption. These indels can induce a frameshift, leading to a premature stop codon and resulting in a truncated amino acid sequence (AS) of the protein (right side).

Article Snippet: Regarding the osteogenic differentiation of hMSCs, 1 × 10 4 cells/cm 2 were maintained in 0.2 mL hMSC growth medium (MSC Growth Medium 2; PromoCell) per cm 2 of tissue culture surface area to obtain a cell monolayer at 100% confluency within 72 h. Duplicate samples were prepared for all experimental setups and time points given.

Techniques: Biomarker Discovery, Sequencing, Disruption, T7EI Assay, CRISPR, Amplification, Nucleic Acid Electrophoresis, Derivative Assay, Control, Mutagenesis, Software, Marker, TA Cloning, Binding Assay, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: Quantification of XYLT1, XYLT2 mRNA expression, and XT-I activity in chondrogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were formed into spheroids and cultured in either standard (control) or chondrogenic differentiation medium (differentiation) for 2 or 7 days. The mRNA levels of ( A ) XYLT1 and ( B ) XYLT2 were assessed at day 2 via qRT-PCR. ( C ) The XT-I enzyme activity per sample volume (nU/volume) was measured at days 2 and 7 using the mass spectrometric XT-I assay. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 (A, B) or n = 2 ( C ) technical replicates per biological replicate, normalized to the WT sample. Mann–Whitney ( A , B ) and Kruskal–Wallis ( C ) test significance levels: not significant (ns), p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****).

Techniques: Expressing, Activity Assay, Cell Culture, Transfection, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: Quantification of XYLT1 , XYLT2 mRNA expression, and XT-I activity in osteogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured post-transfection to 90% confluence, then seeded at 100 cells/mm 2 and grown to 100% confluence. Cultivation continued for 7 or 14 days in either standard (control) or osteogenic differentiation medium (differentiated). The mRNA levels of ( A ) XYLT1 and ( B ) XYLT2 were assessed via qRT-PCR, while ( C ) XT-I enzyme activity (nU/ng) was determined using the mass spectrometric XT-I assay, normalized to DNA content. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 ( A , B ) or n = 2 ( C ) technical replicates per biological replicate, normalized to the WT controls. Kruskal–Wallis test significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Techniques: Expressing, Activity Assay, Cell Culture, Transfection, Control, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: Quantification of XYLT1, XYLT2 mRNA expression, and XT-I activity in adipogenically differentiated WT and KD hMSC cultures. The hMSCs were cultured post-transfection with (KD) or without (WT) the RNP complex until reaching 90% confluency. Cells were seeded at a density of 210 cells/mm 2 and cultured in standard hMSC medium until 100% confluency. Adipogenesis was induced over a period of twenty days by culturing the cells for three cycles of three days in the induction medium, followed by one day in the maintenance medium. Subsequently, cells were cultured in maintenance medium for an additional seven days. The mRNA expression levels of ( A , C ) XYLT1 and ( B , D ) XYLT2 were assessed at day 3 (n = 2 primary cell cultures) and day 20 (n = 1 primary cell culture) via qRT-PCR. ( E ) XT-I enzyme activity (nU/ng) was measured at day 20 (n = 1 primary cell culture) using the mass spectrometric XT-I assay, normalized to DNA content. Data are means ± SEM from n = 3 biological replicates per primary cell culture and n = 3 ( A – D ) or n = 2 ( E ) technical replicates per biological replicate, normalized to the WT sample. Mann–Whitney test significance levels: not significant (ns), (*) p < 0.05, (**) p < 0.01.

Techniques: Expressing, Activity Assay, Cell Culture, Transfection, Quantitative RT-PCR, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: XYLT1 deficiency alters the ECM deposition/PG content of chondrogenically differentiated hMSCs. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were formed into spheroids and cultured in either hMSC standard medium (control) or chondrogenic differentiation medium (differentiation). The pellet cultures were fixed after 14 or 28 days and incubated in a 15% sucrose solution, and 5 μm cryosections were prepared. Cryosections were stained with a 1% alcian blue solution to visualize acidic PG and glycoproteins. Cell nuclei were counterstained blue to purple using Gill’s III hematoxylin solution. Representative images are shown from n = 2 primary cell cultures (indicated with #1, #2), n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Scale bar: 250 μm.

Techniques: Cell Culture, Transfection, Control, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: XYLT1 deficiency affects the collagen deposition of chondrogenically differentiated hMSCs. The hMSCs were cultured to 90% confluence following transfection with (KD) or without (WT) RNP complex. A total of 250,000 cells were pelleted and cultured in either hMSC standard (control) or chondrogenic differentiation (differentiation) medium. The pellet cultures were fixed after 14 or 28 days and incubated in a 15% sucrose solution, and 5 μm cryosections were prepared. Cryosections were stained with Picro-Sirius red solution to visualize collagenous structures in orange-red. Cell nuclei were stained brown-black with acid-resistant Weigert’s iron hematoxylin solution. Representative images are shown from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Scale bar: 250 μm.

Techniques: Cell Culture, Transfection, Control, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: Analysis of osteogenic differentiation in WT and KD hMSCs via qRT-PCR at days 7 and 14. Primary hMSCs were transfected either without (WT) or with (KD) the RNP complex and cultured until reaching 90% confluence. The cells were subcultured at a density of 100 cells/mm 2 , grown until 100% confluence, and then cultivated for 7 or 14 days in either hMSC standard medium (control) or osteogenic differentiation medium (differentiated). The mRNA expression levels of the ( A ) RUNX2 , ( B ) SOX9 , ( C ) OPG , ( D ) COL1A1 , ( E ) BGLAP (osteocalcin), and ( F ) SPP1 (OPN) genes were determined at both day 7 and 14 of osteogenic differentiation. Data are presented as means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate, with normalization to the respective WT control. Kruskal–Wallis significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Techniques: Quantitative RT-PCR, Transfection, Cell Culture, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: Comparison of the mineralization process in WT and KD hMSCs during osteogenesis. The hMSCs were cultured post-transfection without (WT) or with (KD) RNP complex until reaching 90% confluence. Cells were subcultured at a density of 100 cells/mm 2 and, upon reaching 100% confluence, cultured for 7 or 14 days in either hMSC standard medium (control) or osteogenic differentiation medium (differentiated). ( A ) The ALP activity in cell lysates was quantified spectrophotometrically in technical duplicates. The ALP activities (mU/µg) were normalized to the protein content of cell lysates. ( B ) Free inorganic phosphate (P i ) levels (µg/ng) from cell culture supernatants were measured using the ARCHITECT c8000 clinical chemistry analyzer and normalized to the DNA content of the respective cell lysates. ( C ) Calcium deposits were visualized by Alizarin Red S staining using two biological replicates per condition and time point. Representative images of the WT and KD hMSCs cultures on day 7 are shown. Scale bar: 100 µm. ( D ) Osteopontin protein levels in conditioned media were quantified by ELISA, with differentiation controls diluted 1:10 and protein levels normalized to the respective DNA content of the cell lysates. Data are means ± SEM from n = 2 primary cell cultures, n = 3 biological replicates per primary cell culture, and n = 3 technical replicates per biological replicate. Kruskal–Wallis significance levels: p < 0.05 (*), p < 0.01 (**). N/A = not available.

Techniques: Comparison, Cell Culture, Transfection, Control, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: XYLT1 Deficiency of Human Mesenchymal Stem Cells: Impact on Osteogenic, Chondrogenic, and Adipogenic Differentiation

doi: 10.3390/ijms26157363

Figure Lengend Snippet: Quantification of perilipin 1 and leptin protein expression in WT and KD adipogenically differentiated hMSC cultures. The hMSCs were cultured to 90% confluence following transfection without (WT) or with (KD) the RNP complex. Cells were subcultured at a density of 210 cells/mm 2 in standard medium until reaching full confluency. Adipogenic differentiation was conducted over 20 days with cycles of induction and maintenance media, followed by 7 days in maintenance media. Controls remained in maintenance media throughout. ( A ) Cells were fixed for the fluorescence detection of perilipin 1 (magenta) with BODIPY-staining lipid droplets (yellow) and DAPI counterstaining nuclei (cyan). Scale bar = 100 μm. ( B ) Perilipin 1 expression (CTCF) was quantified using ImageJ by analyzing eight images per biological replicate, normalized to total cell number per image. ( C ) Leptin protein levels (pg/ng) in 1:10 diluted cell lysates were measured via ELISA in duplicate, normalized to total DNA. Data are means ± SEM from n = 2 primary cell cultures and n = 3 biological replicates per primary cell culture. Mann–Whitney test significance levels: not significant (ns), p < 0.05 (*).

Techniques: Expressing, Cell Culture, Transfection, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

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