Journal: BioResearch Open Access

Article Title: Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

doi: 10.1089/biores.2012.0252

Figure Lengend Snippet: Phenotype and growth characteristics of human mesenchymal stem cell (hMSCs) and hMSC telomerase reverse transcriptase (TERT) cells. (A) Late-passage [P15(19)] hMSC TERT cells still exhibit the characteristic fibroblastic phenotype of early-passage (P3) hMSCs. (B) Mock-transduced hMSCs and hMSC TERT cells were serial passaged in cell culture, and the population-doubling level (PDL) was calculated at each passage number. At early passages [P2(6)–P7(11)], the PDLs are similar for the two cell lines, but after P8(12), hMSCs proliferate at a slower rate and ceased growth at P16(20) with a maximum PDL of 14. hMSC TERT cells continually proliferate even after PDL 70 [P40(44)].

Article Snippet: For differentiation to adipocytes, hMSCs were treated with three cycles of an hMSC adipogenic induction medium (AIM; Lonza PT-3102B containing h-insulin, l -glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-l-methyl-xanthine [IBMX], and GA-1000) for 3 days and an hMSC adipogenic maintenance medium for 3 days (AMM; Lonza PT-3102A containing h-insulin, l -glutamine, MCGS, and GA-1000), and then further cultured in the AMM until analysis.

Techniques: Reverse Transcription, Cell Culture

Journal: BioResearch Open Access

Article Title: Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

doi: 10.1089/biores.2012.0252

Figure Lengend Snippet: TERT expression in hMSCs and hMSC TERT cells. (A) Relative changes in TERT mRNA expression were determined by real-time reverse transcription (RT)–polymerase chain reaction (PCR) and expressed in relation to 18S levels, and the reference point [mock-transduced hMSC P4(8) levels] using the 2 −ΔΔC(T) method. hMSCs have a low, but detectable, level of TERT mRNA expression at P4(8), but with continued passage TERT expression quickly decreases to nearly undetectable levels even by early-passage P6(10). hMSC TERT cells have significantly high levels of TERT mRNA at all passages, even late passage P16(20). * p <0.05 compared to hMSC levels at each passage. (B) TERT enzymatic activity was also measure at both early [P6(10)] and late [P16(20) and P26(30)] passages. There are significantly higher levels of TERT activity in hMSC TERT cell extracts than in hMSC extracts throughout the time course of the study. * p <0.05 compared to hMSC levels at P6(10).

Article Snippet: For differentiation to adipocytes, hMSCs were treated with three cycles of an hMSC adipogenic induction medium (AIM; Lonza PT-3102B containing h-insulin, l -glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-l-methyl-xanthine [IBMX], and GA-1000) for 3 days and an hMSC adipogenic maintenance medium for 3 days (AMM; Lonza PT-3102A containing h-insulin, l -glutamine, MCGS, and GA-1000), and then further cultured in the AMM until analysis.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Activity Assay

Journal: BioResearch Open Access

Article Title: Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

doi: 10.1089/biores.2012.0252

Figure Lengend Snippet: Expression of p16 INK4a cell cycle regulator in hMSCs and hMSC TERT cells. mRNA levels were quantitated by real-time RT-PCR and normalized to expression levels at P6(10) for each cell line. p16 INK4a levels are significantly increased in hMSCs at later passages [P13(17)], indicating aging of the culture. Levels in hMSC TERT cell lines are unchanged. * p <0.05 compared to P6(10) levels for each cell line.

Article Snippet: For differentiation to adipocytes, hMSCs were treated with three cycles of an hMSC adipogenic induction medium (AIM; Lonza PT-3102B containing h-insulin, l -glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-l-methyl-xanthine [IBMX], and GA-1000) for 3 days and an hMSC adipogenic maintenance medium for 3 days (AMM; Lonza PT-3102A containing h-insulin, l -glutamine, MCGS, and GA-1000), and then further cultured in the AMM until analysis.

Techniques: Expressing, Quantitative RT-PCR

Journal: BioResearch Open Access

Article Title: Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

doi: 10.1089/biores.2012.0252

Figure Lengend Snippet: Enhanced growth characteristics of hMSC TERT cells due to increased cell proliferation and decreased cellular senescence. (A) Cellular proliferation was assayed by measurement of BrdU incorporation during DNA replication, indicating that at both early [P8(12)] and late [P16(20)] passage, the proliferative rate of hMSC TERT cells is significantly higher than that of hMSCs (mock-transduced). * p <0.05 compared to hMSC levels at each passage. The proliferative rate of hMSCs decreases significantly from P8(12) to P16(20), whereas the rate of proliferation of hMSC TERT cells decreases slightly but insignificantly over the same passages. § p <0.05 compared to hMSC levels at P8(12). (B) Cellular senescence was measured by the histochemical stain for β-galactosidase activity. There is a significant increase in β-galactosidase-positive cells in hMSCs after passage P12, whereas the percentage of positive hMSC TERT cells did not increase even after P40(44). * p <0.05 compared to hMSC levels at passage P10. (C) Apoptosis potential was determined by colorimetric determination of DNA fragmentation in hMSCs or hMSC TERT cells. There was no significant difference in the level of apoptosis, although the trend was for lower apoptosis in hMSC TERT cells in comparison to mock-transduced hMSCs.

Article Snippet: For differentiation to adipocytes, hMSCs were treated with three cycles of an hMSC adipogenic induction medium (AIM; Lonza PT-3102B containing h-insulin, l -glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-l-methyl-xanthine [IBMX], and GA-1000) for 3 days and an hMSC adipogenic maintenance medium for 3 days (AMM; Lonza PT-3102A containing h-insulin, l -glutamine, MCGS, and GA-1000), and then further cultured in the AMM until analysis.

Techniques: BrdU Incorporation Assay, Staining, Activity Assay, Comparison

Journal: BioResearch Open Access

Article Title: Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

doi: 10.1089/biores.2012.0252

Figure Lengend Snippet: Chemotaxis toward a medium containing 1% fetal bovine serum (FBS). Data are expressed as a chemotactic index, defined as the number of cells migrating in response to the medium (1% FBS) divided by the number of cells migrating in the negative control (−). hMSC TERT cells [P12(16)] exhibit significantly higher levels of chemotaxis than hMSCs at earlier PDL (P8). * p <0.05 compared to negative control.

Article Snippet: For differentiation to adipocytes, hMSCs were treated with three cycles of an hMSC adipogenic induction medium (AIM; Lonza PT-3102B containing h-insulin, l -glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-l-methyl-xanthine [IBMX], and GA-1000) for 3 days and an hMSC adipogenic maintenance medium for 3 days (AMM; Lonza PT-3102A containing h-insulin, l -glutamine, MCGS, and GA-1000), and then further cultured in the AMM until analysis.

Techniques: Chemotaxis Assay, Negative Control

Journal: BioResearch Open Access

Article Title: Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

doi: 10.1089/biores.2012.0252

Figure Lengend Snippet: Differentiation of hMSC TERT cells toward the osteoblastic and adipogenic lineages. hMSC TERT cells were treated with an osteogenic medium (OGM) for 14 or 28 days. (A) Relative changes in alkaline phosphatase (ALP) expression were determined by real-time RT-PCR. ALP mRNA expression is significantly upregulated at 7, 10, and 14 days. * p <0.05 compared to hMSC TERT levels at day 0. (B) Alizarin Red staining of early- [P5(9)] and late- [P16(20)] passage cells demonstrates that the hMSC TERT cells can undergo terminal differentiation, resulting in the deposition of mineral after OGM treatment for 28 days. (C) Oil Red-O staining of lipid droplets confirms that the hMSC TERT cells can also undergo adipogenic differentiation after induction for 21 days.

Article Snippet: For differentiation to adipocytes, hMSCs were treated with three cycles of an hMSC adipogenic induction medium (AIM; Lonza PT-3102B containing h-insulin, l -glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-l-methyl-xanthine [IBMX], and GA-1000) for 3 days and an hMSC adipogenic maintenance medium for 3 days (AMM; Lonza PT-3102A containing h-insulin, l -glutamine, MCGS, and GA-1000), and then further cultured in the AMM until analysis.

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: International Journal of Molecular Sciences

Article Title: Generation of Mesenchymal Cell Lines Derived from Aged Donors

doi: 10.3390/ijms221910667

Figure Lengend Snippet: Relative expression levels (RELs) of Runx2, Sox9 and Col10A1 in iMSCs spheroids at the beginning of the experiment (t = 0) and after 21 days of chondrogenic induction (t = 21). RELs of each gene were scaled to the REL of the same gene in primary chondrocyte spheroids. Data obtained from iMSC#6, iMSC#9 and iMSC#10 spheroids were employed to calculate the mean values shown.

Article Snippet: Both hanging drop method and pellet cultures were employed to create cell aggregates, which were subsequently incubated in hMSC Chondrogenic Differentiation Medium supplemented with 10 ng/mL of human transforming growth factor β-3 (TGF-β3) (ProSpec-Tany TechnoGene, Rejovot, Israel) for 21 days.

Techniques: Expressing

Journal: Biotechnology and Bioengineering

Article Title: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

doi: 10.1002/bit.25582

Figure Lengend Snippet: Nutrient and metabolite flux of hMSC expansion on microcarriers. Glucose, lactate and ammonia concentrations in FBS‐containing medium (A) and serum‐free medium (B). Total protein (C) and lactate dehydrogenase concentration (D) are shown for FBS‐containing and serum‐free medium. Data shows mean ± SD, n = 3.

Article Snippet: The hMSC differentiation was induced using PRIME‐XV TM hMSC Differentiation Medium (Irvine Scientific, USA) as per manufacturer's instructions.

Techniques: Concentration Assay

Journal: Biotechnology and Bioengineering

Article Title: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

doi: 10.1002/bit.25582

Figure Lengend Snippet: Post‐harvest hMSC quality compared to pre‐expansion demonstrating retention of key attributes, showing (A) specific growth rate, (B) colony forming efficiency, (C) mean cell diameter and (D) forward/side scatter of cell populations confirming difference in mean cell diameter.

Article Snippet: The hMSC differentiation was induced using PRIME‐XV TM hMSC Differentiation Medium (Irvine Scientific, USA) as per manufacturer's instructions.

Techniques:

Journal: Biotechnology and Bioengineering

Article Title: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

doi: 10.1002/bit.25582

Figure Lengend Snippet: Post‐expansion harvest of hMSCs from microcarriers showing successful hMSC detachment from microcarriers (A). Post‐harvest viability shows high number of intact hMSCs for FBS‐containing medium and serum‐free medium. Data shows mean ± SD, n = 3.

Article Snippet: The hMSC differentiation was induced using PRIME‐XV TM hMSC Differentiation Medium (Irvine Scientific, USA) as per manufacturer's instructions.

Techniques:

Journal: Biotechnology and Bioengineering

Article Title: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

doi: 10.1002/bit.25582

Figure Lengend Snippet: Post‐harvest hMSC characterisation. (A) pre‐expansion and (B) post‐harvest hMSC morphology in FBS‐containing medium. (C) pre‐expansion and (D) post‐harvest hMSC morphology in serum‐free medium. Tri‐lineage differentiation of hMSCs showing (E) osteogenic, (F) adipogenic and (G) chondrogenic potential post‐harvest in serum‐free medium. Multiparameter flow cytometry showing dual gating of CD73, 90, 105, 34 and HLA‐DR for hMSCs post‐harvest from serum‐free microcarrier culture (H).

Article Snippet: The hMSC differentiation was induced using PRIME‐XV TM hMSC Differentiation Medium (Irvine Scientific, USA) as per manufacturer's instructions.

Techniques: Flow Cytometry

Journal: Biotechnology and Bioengineering

Article Title: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

doi: 10.1002/bit.25582

Figure Lengend Snippet: Post‐thaw hMSC recovery following serum‐free cryopreservation, showing (A) post‐thaw recovery and 3 h cell attachment based on PI exclusion. Post‐thaw hMSC outgrowth (B) following serum‐free cryopreservation. Data shows mean ± SD (n = 3).

Article Snippet: The hMSC differentiation was induced using PRIME‐XV TM hMSC Differentiation Medium (Irvine Scientific, USA) as per manufacturer's instructions.

Techniques: Cell Attachment Assay

Journal: Biotechnology and Bioengineering

Article Title: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

doi: 10.1002/bit.25582

Figure Lengend Snippet: Post‐thaw hMSC recovery following serum‐free cryopreservation, demonstrating formation of F‐Actin cytoskeleton (A) 3 h, (B) 24 hours post thaw (C) 3 h post‐passage control and (D) 24 hours post‐passage control. Phase contrast images show day 2 hMSC morphology post‐thaw (E) and post passage control (F). Scale bar = 250 μm.

Article Snippet: The hMSC differentiation was induced using PRIME‐XV TM hMSC Differentiation Medium (Irvine Scientific, USA) as per manufacturer's instructions.

Techniques: Control

Journal: Micromachines

Article Title: 3D Printed Wavy Scaffolds Enhance Mesenchymal Stem Cell Osteogenesis

doi: 10.3390/mi11010031

Figure Lengend Snippet: ( A ) Optical microscopy images of the hMSCs stained for alizarin red (red) after culture in osteogenic induction media for 21 days. Scale bars are 200 microns. ( B ) Alizarin red concentration indicating calcium deposition at Day 21. (* p < 0.15, ** p < 0.05, for n = 3).

Article Snippet: For osteogenic differentiation studies, growth media was replaced with osteogenic induction media (hMSC osteogenic differentiation medium BulletKitTM, Lonza, Basel, Switzerland) at Day 7, and cells were cultured for an additional 14 days.

Techniques: Microscopy, Staining, Concentration Assay

Journal: Micromachines

Article Title: 3D Printed Wavy Scaffolds Enhance Mesenchymal Stem Cell Osteogenesis

doi: 10.3390/mi11010031

Figure Lengend Snippet: Multiphoton confocal images for hMSCs that were cultured in osteogenic induction media for 14 ( top row) and 21 days ( bottom row). Cells were immunostained for osteocalcin (green) and stained for F-actin (red) and cell nuclei (blue). Scale bars are 200 microns.

Article Snippet: For osteogenic differentiation studies, growth media was replaced with osteogenic induction media (hMSC osteogenic differentiation medium BulletKitTM, Lonza, Basel, Switzerland) at Day 7, and cells were cultured for an additional 14 days.

Techniques: Cell Culture, Staining