neonatal foreskin fibroblasts  (ATCC)

Journal: Scientific Reports

Article Title: Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

doi: 10.1038/srep44534

Figure Lengend Snippet: ( A ) The experimental scheme for efficient derivation of iMSCs from dermal fibroblasts with 6 chemicals with or without LIF, TGF-β, and bFGF. Expanded iMSCs with clonal expansion ability (clonogenicity) can be further differentiated into different lineages (multipotency) or treat sepsis-induced acute lung injury in the mouse model. ( B ) The representative flow cytometry analysis of human dermal fibroblasts CRL2097, iMSCs, and BMMSCs with MSC functional markers SSEA-4 and PODXL. SSEA-4 and PODXL were abundantly expressed in iMSCs and BMMSCs, but not in fibroblasts. ( C ) Clonogenicity of fibroblasts, iMSCs, and BMMSCs. By a 96-well colony formation assay, quantitation of CFU-F in fibroblasts (1 per 112 cells), iMSCs (1 per 7 cells), and BMMSCs (1 per 22 cells) was obtained by the negative linear relationship between the number of seeded cells and the percentage of wells with no colonies. ( D ) Flow cytometry analysis of OCT4 protein expression in human fibroblasts, iMSCs, BMMSCs, and human embryonic stem cells (hESCs). The pluripotent marker OCT4 was at a basal level in fibroblasts (CRL2097), slightly up-regulated in iMSCs and BMMSCs, and highly expressed in hESCs. ( E ) The principal component analysis of the expression of stemness genes in three different dermal fibroblasts (CRL2097, DF440547, and DF443480), iMSCs induced from the three different fibroblasts, and two independent sources of BMMSCs (BMMSC_1: primary human bone marrow MSCs used throughout this study; BMMSC_2: publicly available gene expression data for human BMMSCs with accession number GSM1533333). Expression probes were functionally annotated with Gene Ontology (GO) and selected by text-mining for the term “stem cell maintenance” in R (418 probes). Principal component 1 accounts for 40%, principal component 2 accounts for 19%, and principal component 3 accounts for 14% of the variation in the dataset. The clustering of iMSCs derived from three different fibroblast sources between two different BMMSCs suggests the robust efficacy of the cocktail.

Article Snippet: Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Flow Cytometry, Functional Assay, Colony Assay, Quantitation Assay, Expressing, Marker, Gene Expression, Derivative Assay

Journal: Scientific Reports

Article Title: Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

doi: 10.1038/srep44534

Figure Lengend Snippet: ( A ) To detect early osteogenesis, fibroblasts (CRL2097), iMSCs derived from CRL2097 (iMSCs), and BMMSCs were cultured in osteogenic induction medium for 10 days, and the alkaline phosphatase (ALP) activity assay was performed. ( B ) To detect late osteogenesis, Alizarin Red S staining (ARS) was performed at day 21. ( C ) To detect adipogenesis, fibroblasts CRL2097, iMSCs, and BMMSCs were cultured in adipogenic induction medium for 21 days and then stained with Oil Red O. Scale bar: 50 μm. ( D ) The quantification results of the ALP activity, the Alizarin Red S assay, and the Oil Red O staining (n = 6). **** p < 0.0001. ( E ) The lacunae structure (Hematoxylin and eosin staining, HE) and proteoglycans of cartilage (Alcian Blue staining) were examined to evaluate the ability of iMSCs to differentiate into chondrocytes at day 21. The lacunae are marked by the yellow arrow. Scale bar, 50 μm.

Article Snippet: Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Derivative Assay, Cell Culture, ALP Activity Assay, Staining, Activity Assay

Journal: Scientific Reports

Article Title: Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

doi: 10.1038/srep44534

Figure Lengend Snippet: ( A ) The experimental scheme for inducing acute lung injury in mice using intratracheal injections of LPS. ( B ) iMSCs and BMMSCs significantly ameliorate lung inflammation. Representative lung histology at 48 h after LPS-induced acute lung injury. Scale bar: 100 μm. ( C ) iMSCs, like BMMSCs, efficiently improve the survival rate of LPS-induced acute lung injury. The results are expressed as a percentage of survival (n = 10–12 per group). * p < 0.05, LPS + PBS V.S. (PBS, LPS + iMSCs, or LPS + BMMSCs). # p < 0.05, LPS + Fibroblasts vs. (PBS, LPS + iMSCs, or LPS + BMMSCs). ( D ) The injury score of LPS-induced acute lung injury and the quantification of the histology at 48 h after LPS-induced acute lung injury. All sections were quantified after digital slide scanning of the whole slide. n = 10–12 each group. The details of calculation were described in method. The injury score used the following criteria: 0, no injury; 1, 25% injury in the field; 2, 50% injury in the field; 3, 75% injury in the field; and 4, diffuse lung injury. * p < 0.05.

Article Snippet: Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques:

Journal: Scientific Reports

Article Title: Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

doi: 10.1038/srep44534

Figure Lengend Snippet: After 4 hours, LPS-treated mice were intratracheal instilled with PBS, fibroblasts, iMSCs, or BMMSCs. The expression levels of indicated proinflammatory cytokines in BALF were detected by Multiplex bead array assay. The levels of IL-1β, TNF-α, and TNF-β in BALF were quantified in three independent experiments. Data shows in means with ± SEM. * p < 0.05; ** p < 0.01; n.s.: not significant.

Article Snippet: Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Expressing, Multiplex Assay

Journal: Scientific Reports

Article Title: Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

doi: 10.1038/srep44534

Figure Lengend Snippet: ( A ) The optimization of the cocktail compositions for the conversion of human fibroblasts into iMSCs. The chemical cocktail contained at most six chemical inhibitors: JNKi (SP600125, 10 μM), p38i (SB202190, 10 μM), PKCi (Go6983, 5 μM), ROCKi (Y-27632, 5 μM), ERK1/2i (PD0325901, 1 μM), and GSK3βi (CHIR99021, 3 μM). The SSEA-4 high PODXL high population was quantified to measure the iMSC conversion efficacy at day 6. Three compounds, SP600125, SB202190, and Go6983, were sufficient for the conversion of human fibroblasts to iMSCs with low efficiency (condition 2). The removal of Y-27632, PD0325901, or CHIR99021 reduced the efficacy of iMSC production (conditions 4, 9, and 10). The cocktail containing all six inhibitors (condition 8) resulted in the most efficacious conversion, with an efficacy comparable to the chemical cocktail (6C+3GF) (condition 11, 6 chemicals with three cytokines). BMMSCs served as the positive control, and fibroblasts served as the negative control. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s.: not significant. ( B ) The iMSCs derived from fibroblasts CRL2097 with only 6 chemicals exhibit osteogenesis abilities comparable to those of BMMSCs. Fibroblasts, iMSCs derived with 6 chemicals (6 C), and BMMSCs were cultured in osteoblast-induction medium for 21 days, and then, they were assayed by Alizarin Red staining (ARS) (left panel). The dye was extracted, and ARS was quantified by measuring the optical density (O.D.) at 550 nm (right panel) (n = 6). **** p < 0.0001. ( C ) The iMSCs derived from fibroblasts CRL2097 with only 6 chemicals exhibit adipogenesis abilities comparable to those of BMMSCs. Indicated fibroblasts, iMSCs, and BMMSCs were cultured in adipocyte induction medium for 21 days, and the lipid drops were then stained with Oil Red O (left panel). Scale bar: 50 μm. The dye was extracted, and Oil Red O staining was quantified by measuring the O.D. at 530 nm (right panel) (n = 6). **** p < 0.0001.

Article Snippet: Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Positive Control, Negative Control, Derivative Assay, Cell Culture, Staining

Journal: Scientific Reports

Article Title: Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

doi: 10.1038/srep44534

Figure Lengend Snippet: The chemical cocktail with six chemical inhibitors with or without three growth factors efficiently and robustly generated iMSCs from primary dermal fibroblasts in six days. iMSCs are multipotent and can further differentiate into osteoblasts (bone lineage), adipocytes (fat lineage), and chondrocytes (cartilage lineage). iMSCs also markedly increased the clonogenicity and decreased the fatality of endotoxin-induced acute lung injury in a mouse model.

Article Snippet: Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Generated