Journal: Cell Transplantation

Article Title: Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells

doi: 10.1177/0963689720965529

Figure Lengend Snippet: Generation of B2M knockout hP-iPSCs with CRISPR/Cas9 and double-color selection. (A) Schematic of a CRISPR/Cas9 system targeting B2M EX1 with two selection donor templates for homologous recombination. The system was used for genetic modification in hP-iPSCs by electroporation. Arrows showed the binding sites of PCR primers for genotyping. (B) Representative images of B2M biallelic knockout hP-iPSC single-cell clone. The scale bar was 200 μm, and the exposure time was 1 s for fluorescence. (C) PCR analysis of WT and HDR alleles of the B2M gene from B2M monoallelic and biallelic knockout hP-iPSC single-cell clones. WT hP-iPSCs were included as a control. (D) Western blot analysis of B2M expression in B2M monoallelic and biallelic knockout hP-iPSC single-cell clones. Sample cells were treated by interferon-γ for 48 h before analysis. WT clone was used as a control, and β-actin was detected as housekeeping expression. (E) Representative flow cytometry diagrams of surface B2M and HLA-A, B, C expression on WT, B2M monoallelic and biallelic knockout hP-iPSC single-cell clones. CRISPR/Cas9: clustered regulatory interspaced short palindromic repeat-associated Cas9 endonuclease; EX1: exon 1; HDR: homology-direct recombination; HLA: human leukocyte antigen; hP-iPSCs: human peripheral blood mononuclear cell-derived induced pluripotent stem cells; PCR: polymerase chain reaction; WT: wild type.

Article Snippet: Human PBMC-derived iPSCs were reprogrammed from healthy donor’s PBMCs using Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. hP-iPSCs were selected as single-cell clones and characterized as described previously . hP-iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel hESC-qualified Matrix (BD Biosciences, Franklin Lakes, NJ, USA) coated plates.

Techniques: Knock-Out, CRISPR, Selection, Homologous Recombination, Modification, Electroporation, Binding Assay, Fluorescence, Clone Assay, Control, Western Blot, Expressing, Flow Cytometry, Derivative Assay, Polymerase Chain Reaction

Journal: Cell Transplantation

Article Title: Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells

doi: 10.1177/0963689720965529

Figure Lengend Snippet: One-step generation of B2MKO hP-iPSCs by CRISPR/Cas9 technology. ( A ) Representative flow cytometry diagrams of surface B2M and HLA-A, B, C expression on WT, B2MKO #3 and #8 hP-iPSC single-cell clones. (B) Sanger Sequencing analysis of B2M exon1 in B2MKO #3 and #8 hP-iPSC single-cell clones. CRISPR targeting sequences were shown in orange, and the target site in the B2M gene was shown in green. The insertion was marked by red, while deletion was marked by “-”. The genotypes of B2MKO #3 and #8 hP-iPSC single-cell clones were summarized. B2MKO: B2M knockout; CRISPR/Cas9: clustered regulatory interspaced short palindromic repeat-associated Cas9 endonuclease; HLA: human leukocyte antigen; hP-iPSCs: human peripheral blood mononuclear cell-derived induced pluripotent stem cells; WT: wild type.

Article Snippet: Human PBMC-derived iPSCs were reprogrammed from healthy donor’s PBMCs using Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. hP-iPSCs were selected as single-cell clones and characterized as described previously . hP-iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel hESC-qualified Matrix (BD Biosciences, Franklin Lakes, NJ, USA) coated plates.

Techniques: CRISPR, Flow Cytometry, Expressing, Clone Assay, Sequencing, Knock-Out, Derivative Assay

Journal: Cell Transplantation

Article Title: Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells

doi: 10.1177/0963689720965529

Figure Lengend Snippet: Pluripotency of B2MKO hP-iPSCs. (A) RT-PCR analysis of pluripotent marker Oct4, Sox2, and Nanog expression in WT and B2MKO hP-iPSC single-cell clones. The expression of β-actin was included as a housekeeping control. (B) Representative images of EBs derived from WT and B2MKO hP-iPSC single-cell clones. The scale bar was 200 μm. (C) RT-PCR analysis of markers’ expression of three germ layers in WT and B2MKO hP-iPSC-derived EBs. Pax6 was detected for ectoderm; MHC-α was detected for mesoderm, and AFP was detected for endoderm. The expression of β-actin was included as a housekeeping control. (D) Immunostaining of three germ layer markers in WT and B2MKO hP-iPSC-derived EBs. Beta-III-tubulin (TUJ1) was stained for ectoderm in blue color; SMA was stained for mesoderm in red color, and AFP was stained for endoderm in green color. The scale bar was 500 μm. AFP: alpha-fetoprotein; B2MKO: B2M knockout; EB: embryoid bodies; hP-iPSCs: human peripheral blood mononuclear cell-derived induced pluripotent stem cells; SMA: smooth muscle actin; RT-PCR: reverse transcription-polymerase chain reaction; WT: wild type.

Article Snippet: Human PBMC-derived iPSCs were reprogrammed from healthy donor’s PBMCs using Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. hP-iPSCs were selected as single-cell clones and characterized as described previously . hP-iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel hESC-qualified Matrix (BD Biosciences, Franklin Lakes, NJ, USA) coated plates.

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Clone Assay, Control, Derivative Assay, Immunostaining, Staining, Knock-Out, Reverse Transcription, Polymerase Chain Reaction

Journal: Cell Transplantation

Article Title: Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells

doi: 10.1177/0963689720965529

Figure Lengend Snippet: Generation of mesenchymal stromal cells from B2MKO hP-iPSCs. (A) Representative images of WT and B2MKO hP-iPSC-derived MSCs. The scale bar was 200 μm. (B) Representative flow cytometry diagrams of surface B2M and HLA-A, B, C expression on WT and B2MKO iMSCs. (C) Phenotyping of WT and B2MKO iMSCs by surface markers. The expression of MSC negative markers (CD14, CD24, CD34, CD45, and HLA-DR) and MSC positive markers (CD29, CD44, CD73, CD90, CD105, and CD166) was shown as representative flow cytometry diagrams. B2MKO: B2M knockout; HLA: human leukocyte antigen; hP-iPSCs: human peripheral blood mononuclear cells-derived induced pluripotent stem cells; MSCs: mesenchymal stromal cells; WT: wild type.

Article Snippet: Human PBMC-derived iPSCs were reprogrammed from healthy donor’s PBMCs using Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. hP-iPSCs were selected as single-cell clones and characterized as described previously . hP-iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel hESC-qualified Matrix (BD Biosciences, Franklin Lakes, NJ, USA) coated plates.

Techniques: Derivative Assay, Flow Cytometry, Expressing, Knock-Out

Journal: Cell Transplantation

Article Title: Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells

doi: 10.1177/0963689720965529

Figure Lengend Snippet: Multipotency of B2MKO hP-iPSC-derived MSCs. (A) Adipogenesis, (B) osteogenesis, and (C) chondrogenesis of WT and B2MKO iMSCs. Lipid content was stained in red by Oil Red O for adipocytes; calcific deposition was stained in red by Alizarin Red S for osteocytes, and acidic polysaccharides were stained in blue by Alcian blue 8GX for chondrocytes. The scale bar for all the images was 80 μm. Expression of specific markers was detected by RT-PCR for both WT and B2MKO iMSCs and differentiated cells. (D) LPL and PPAR-γ were detected for adipogenesis. (E) Bone relevant ALP and OCN were detected for osteogenesis. (F) ACAN and COL2a were detected for chondrogenesis. ACAN: aggrecan; ALP: alkaline phosphatase; B2MKO: B2M knockout; COL2a: collagen type II alpha 1; hP-iPSCs: human peripheral blood mononuclear cell-derived induced pluripotent stem cells; LPL: lipoprotein lipase; MSC: mesenchymal stromal cells; OCN: osteocalcin; PPAR-γ: peroxisome proliferator-activated receptor-gamma; RT-PCR: reverse transcription-polymerase chain reaction; WT: wild type.

Article Snippet: Human PBMC-derived iPSCs were reprogrammed from healthy donor’s PBMCs using Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. hP-iPSCs were selected as single-cell clones and characterized as described previously . hP-iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel hESC-qualified Matrix (BD Biosciences, Franklin Lakes, NJ, USA) coated plates.

Techniques: Derivative Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Reverse Transcription, Polymerase Chain Reaction

Journal: Cell Transplantation

Article Title: Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells

doi: 10.1177/0963689720965529

Figure Lengend Snippet: Immunosuppressive property of B2MKO hP-iPSC-derived MSCs. (A) Tracking proliferation of stimulated PBMCs in the presence or absence of iMSCs by Far Red staining. iMSCs were introduced in a PBMCs/iMSCs ratio of 2:1. Allogeneic PBMCs were stimulated by 50 ng/ml OKT-3 and the dilution of Far Red staining was gated. Representative set of flow cytometry histograms from three individual donors was shown. (B) Allogeneic PBMCs proliferation was summarized according to the percentage of relative proliferation. The dilution of Far Red in stimulated PBMCs without iMSCs was normalized as 100% in relative proliferation, while the dilution of Far Red in PBMCs without OKT-3 was 0% in relative proliferation. Bars show the mean ± SD of relative proliferation in three independent experiments with individual donors. ** P < 0.01; * P < 0.05. B2MKO: B2M knockout; hP-iPSCs: human peripheral blood mononuclear cell-derived induced pluripotent stem cells; MSC: mesenchymal stromal cells; PMBC: peripheral blood mononuclear cell; RT-PCR: reverse transcription-polymerase chain reaction.

Article Snippet: Human PBMC-derived iPSCs were reprogrammed from healthy donor’s PBMCs using Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. hP-iPSCs were selected as single-cell clones and characterized as described previously . hP-iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel hESC-qualified Matrix (BD Biosciences, Franklin Lakes, NJ, USA) coated plates.

Techniques: Derivative Assay, Staining, Flow Cytometry, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction

Journal: Cell Transplantation

Article Title: Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells

doi: 10.1177/0963689720965529

Figure Lengend Snippet: Hypoimmunogenicity of B2MKO hP-iPSC-derived mesenchymal stromal cells. (A, C) Phenotyping of iMSC-primed PBMCs was shown as flow cytometry diagrams for CD3, CD56, and CD8 expression. (B, D) Immunogenicity of both WT and B2MKO iMSCs when they were challenged by iMSC-primed PBMCs. The immunogenicity was examined with DELFIA EuTDA cytotoxicity assays (2 h Eu-ligand release) as target cells challenged by iMSC-primed PBMCs. (E) The susceptibility of WT and B2MKO iMSC to NK lysis. Primary NK cells were used as effectors to target WT and B2MKO iMSCs in DELFIA EuTDA cytotoxicity assays (2 h Eu-ligand release). For cell cytotoxicity assays, three independent assays from three individual donors were performed. Shown are percentage lysis of target cells at varying E:T ratios in one representative experiment (mean ± SD of triplicate samples). *** P < 0.001. B2MKO: B2M knockout; hP-iPSCs, human peripheral blood mononuclear cell-derived induced pluripotent stem cells; MSC: mesenchymal stromal cells; NK: natural killer; PMBC: peripheral blood mononuclear cell; WT: wild type.

Article Snippet: Human PBMC-derived iPSCs were reprogrammed from healthy donor’s PBMCs using Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. hP-iPSCs were selected as single-cell clones and characterized as described previously . hP-iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel hESC-qualified Matrix (BD Biosciences, Franklin Lakes, NJ, USA) coated plates.

Techniques: Derivative Assay, Flow Cytometry, Expressing, Immunopeptidomics, Lysis, Knock-Out

Journal: Cell Proliferation

Article Title: Manufacturing clinical‐grade human induced pluripotent stem cell‐derived beta cells for diabetes treatment

doi: 10.1111/cpr.13232

Figure Lengend Snippet: Comparison of Sendai virus, episomal and mRNA reprogramming methods for clinical and commercial use

Article Snippet: Source companies with rights to reprogramming kits (non‐exhaustive list) , Thermo Fisher Scientific (CytoTune‐iPS Sendai Reprogramming Kit) , Thermo Fisher Scientific (Epi5 Episomal iPSC Reprogramming Kit) Lonza (Lonza L7 hiPSC Reprogramming and hPSC Culture System) Alstem (Episomal iPSC Reprogramming Kit) Creative Bioarray (QualiStem Episomal iPSC Reprogramming Kit) , Reprocell (Stemgent StemRNA 3rd Gen Reprogramming Kit) Creative Bioarray (QualiStem RNA iPSC Reprogramming Kit) Stem Cell Technologies (ReproRNA‐OKSGM) Merck (Simplicon RNA Reprogramming Kit).

Techniques: Comparison, Virus, Plasmid Preparation, Transfection

Journal: Cell Proliferation

Article Title: Manufacturing clinical‐grade human induced pluripotent stem cell‐derived beta cells for diabetes treatment

doi: 10.1111/cpr.13232

Figure Lengend Snippet: Non‐exhaustive list of regulatory authorities and stem cell organizations involved in stem cell therapies

Article Snippet: Source companies with rights to reprogramming kits (non‐exhaustive list) , Thermo Fisher Scientific (CytoTune‐iPS Sendai Reprogramming Kit) , Thermo Fisher Scientific (Epi5 Episomal iPSC Reprogramming Kit) Lonza (Lonza L7 hiPSC Reprogramming and hPSC Culture System) Alstem (Episomal iPSC Reprogramming Kit) Creative Bioarray (QualiStem Episomal iPSC Reprogramming Kit) , Reprocell (Stemgent StemRNA 3rd Gen Reprogramming Kit) Creative Bioarray (QualiStem RNA iPSC Reprogramming Kit) Stem Cell Technologies (ReproRNA‐OKSGM) Merck (Simplicon RNA Reprogramming Kit).

Techniques: