- Email: [email protected]
Generation of four integration-free iPSC lines from related human donors
Stem Cell Research 41 (2019) 101615
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Generation of four integration-free iPSC lines from related human donors ⁎
T
Anja Patricia Ramme , Daniel Faust, Leopold Koenig, Uwe Marx TissUse GmbH, Oudenarder Str. 16, 13347 Berlin, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: iPSCs Induced pluripotent stem cells Reprogramming Differentiation Human
Four integration-free iPSC lines were generated by reprogramming peripheral blood mononuclear cells with episomal vectors. All four human donors (two male and two female donors) belong to one Caucasian family within three different generations with the age between 19–82 years. Additionally, all iPSC lines are approved for commercial use by donor consent. Those iPSC lines offer the opportunity to study the influence of affiliation within one family. In future, more iPSCs lines of many more family members can be created to understand the effects of relatives with different ages on the reprogramming into iPSCs and differentiation into specific cell types.
Resource Table:
Cell line repository/bank Ethical approval
Unique stem cell lines identifier
TISSUi001-A TISSUi002-A TISSUi003-A TISSUi005-A Alternative names of stem cell li- TISSUi001-A: StemUse101, HUMIMIC101, nes SU101 TISSUi002-A: StemUse102, HUMIMIC102, SU102 TISSUi003-A: StemUse103, HUMIMIC103, SU103 TISSUi005-A: StemUse105, HUMIMIC105, SU105 Institution TissUse GmbH, Berlin, Germany Contact information of distributor [email protected], Anja Ramme Type of cell lines iPSCs Origin human Cell Source PBMCs Clonality Clonal Method of reprogramming episomes (Epi5 Kit, Thermo Fisher A15960) Multiline rationale One Caucasian family Gene modification No Type of modification N/A Associated disease 3 healthy donors, one leukemia patient Gene/locus N/A Method of modification N/A Name of transgene or resistance N/A Inducible/constitutive system N/A Date archived/stock date deposited in repository: StemUse101 06/2017 StemUse102 08/2017 StemUse103 09/2017 StemUse105 11/2017
⁎
Human Pluripotent Stem Cell Registry (hPSCreg): https://hpscreg.eu/search?q=TISSUi Ethikkomission der Ärztekammer Berlin; Eth 25/ 16
1. Resource utility There is a lack of iPSC lines with donor consent for commercial use, since many iPSC lines are only available for research. We generated four integration-free iPSC lines with donor consent for commercial use from one family to study the influence of age and gender on the reprogramming into iPSCs and differentiation into specific cell types. 2. Resource details Two male humans and two female humans from one Caucasian family (family tree in supplements Fig. S 2) with the age between 19 and 82 donated blood for iPSC generation. The integration-free reprogramming was performed by transfection with episomal vectors (Epi5 Kit, ThermoFisher A15960). The StemUse105 iPSC line was reprogrammed from a patient with leukemia, all three other lines were derived from healthy donors. Bright-field images of all four lines show colony morphology with tightly packed cells with high nucleus to cytoplasm ratio (Fig. 1A–D, I and II, scale 100 μm). The pluripotency state was characterized by positive immunofluorescence staining of NANOG, SSEA5, OCT3/4 and negative staining of α-Actin in all four iPSC lines (Fig. 1A–D, III–V, scale 100 μm). Flow cytometry staining reveals more than 95% of the cell population are positive for TRA-1-60 and negative for SSEA1, 98% are positive for SSEA5 and TRA-1-60, 92% are positive
Corresponding author. E-mail address: [email protected] (A.P. Ramme).
https://doi.org/10.1016/j.scr.2019.101615 Received 28 August 2019; Received in revised form 27 September 2019; Accepted 8 October 2019 Available online 19 October 2019 1873-5061/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Stem Cell Research 41 (2019) 101615
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Fig. 1. Characterization of the hiPSC lines TISSUi001-A (A), TISSUi002-A (B), TISSUi003-A (C) and TISSUi005-A (D). Phase contrast images of respective iPSC lines (I and II) and immunofluorescent analysis of pluripotency markers (NANOG, SSEA5, OCT3/4 and negative staining of α-Actin) (III IV). (E) Quantitative analysis by flow cytometry of the pluripotency markers (TRA-1-60+/ SSEA1, SSEA5+/ TRA-1-60+; OCT3/4+/ SOX2+ and NANOG+), definitive endoderm marker (CXCR4 and FOXA2) and mesoderm marker (MHC and cTnT).
bioinformatics assay PluriTest™ (Supplementary data Fig. S 1A). We performed monolayer-based directed differentiation of all four iPSC lines into ectoderm cells (neuronal cells), endoderm cells and
for OCT3/4 and SOX2 and 92% are positive for NANOG in all four iPSC lines (Fig. 1E and supplements Fig. S 3–S 6). The pluripotency of all four lines was further proved with the transcriptomic data based 2
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Table 1 Summary of lines. iPSC line names
Abbreviation in figures
Gender
Age
Ethnicity
Genotype of locus
Disease
StemUse101 StemUse102 StemUse103 StemUse105
SU101 SU102 SU103 SU105
Male Female Female Male
52 45 19 82
Caucasian Caucasian Caucasian Caucasian
N/A N/A N/A N/A
N/A N/A N/A Leukemia
3. Materials and methods
mesoderm cells (cardiomyocytes). Neuronal differentiation was shown by positive staining for the early neuronal marker TUBB3 and the neuronal stem cell marker PAX6 in all four iPSC lines (Fig. 1A–D, VI, scale 100 μm). Definitive endoderm differentiation was performed using a commercial available kit and resulted into more than 91% CXCR4 positive cells and more than 77% FOXA2 positive cells (Fig. 1E and supplements Fig. S 7–S 10). All four iPSC lines differentiated into beating cardiomyocytes shown by the expression of myosin heavy chain (MHC) and cardiac troponin T (cTnT) (between 4% and 74% depending on the iPSC line) (Fig. 1E and supplements Fig. S 11–S 14). The KaryoStat™ assay was performed by Life Technologies Corporation and revealed no chromosomal aberrations in all four iPSC lines (Supplementary data Fig. S 1B). However, balanced translocations cannot be identified by the KaryoStat™ assay. The analysis of 15 short tandem repeats and X loci proved the identity of all four iPSC lines to the corresponding blood sample (data available with the authors). All four iPSC lines were tested negative for mycoplasma (Supplementary data Fig. S 15 and S 16) and bacterial contaminations. All data for the characterization of the lines are summarized in Tables 1–3. In the future all StemUse lines will be further differentiated in different cell types. Afterwards, those different cell types will be co-cultured in Multi-Organ-Chips (TissUse GmbH, Berlin, Germany) to study the interaction of organ models. We already showed this approach from the StemUse101 line. Four different organ models - intestine, liver, renal and neuronal models - from the StemUse101 line were differentiated and co-cultured in the four-organ-chip (HUMIMIC Chip4) and maintained for up to 14 days in a common growth factor deprived medium (Ramme et al., 2019). Additionally the StemUse101 line was used for 3D neuronal differentiation in a bioreactor system (Koenig et al., 2018).
3.1. iPSC culture Cell culture plates and components were purchased from Corning U.S. and cultures were incubated at 37 °C and 5% CO2, unless otherwise stated. The human iPSC lines HUMIMIC StemUse101, StemUse102, StemUse103 and StemUse105 (TissUse GmbH, Berlin, Germany) were derived from peripheral blood mononuclear cells. Reprogramming was performed by the service provider Phenocell SAS (Grasse, France) by transfection with episomal vectors (Epi5 Kit, Thermo Fisher A15960). The iPSCs were maintained in feeder-free conditions in StemMACS iPSBrew XF (Miltenyi Biotec) on growth factor-reduced (GFR) Matrigel® (1:100 diluted in KO/DMEM F12; Thermo Fisher) on cell culture treated dishes. The iPSCs were passaged every five to seven days using Accutase®, 4,000–6,000 cells/cm² were seeded in culture medium with 10 μM Rock Inhibitor (RI) Y-27632 (Cayman). StemMACS iPS-Brew XF medium without RI was renewed after 48 h, following a daily medium exchange.
3.2. Direct differentiation into definitive endoderm All four iPSC lines were differentiated into the definitive endoderm (DE) using the STEMdiff™ DE Kit (TeSR™-E8™ Optimized, STEMCELL Technologies), according to the manufacturer's instructions, with minor modifications. The iPSCs were split with Accutase and seeded with 33,000 cells/cm² cells on GFR Matrigel in iPSC medium supplemented with 10 μM RI and STEMdiff™ DE TeSR™-E8™ Supplement (1:20). Following medium changes were performed according to the manufacturer's instructions.
Table 2 Characterization and validation. Classification
Test
Result
Data
Morphology Phenotype
Photography Qualitative analysis (Immunocytochemistry) Quantitative analysis (Flow cytometry)
normal Positive for pluripotency markers: OCT3/4, NANOG, SOX2 and SSEA5 TRA-1-60+/ SSEA1-: 95% SSEA5+/ TRA-1-60+: 98% OCT3/4+/ SOX2+: 92% NANOG+: 92% No chromosomal aberrations were found in SU101, SU102, SU103 and SU105 when comparing against the reference dataset. DNA Profiling performed by STR analysis 16 sited tested and all matched N/A N/A Negative
Fig. 1 (A–D, I and II) Fig. 1 (A–D, III–V)
Genotype
KaryoStat™ assay
Identity
STR analysis
Mutation analysis
Sequencing Southern Blot OR WGS MycoAlert® Mycoplasma Detection Kit (LT07-218) and PCR Directed differentiation
Microbiology and virology Differentiation potential
Donor screening Genotype additional info
HIV 1 + 2 Hepatitis B, Hepatitis C Blood group genotyping HLA tissue typing
Definitive endoderm marker: CXCR4 and FOXA2 Mesoderm marker: MHC and cTnT Ectoderm marker: TUBB3 and PAX6 Negative for all four donors N/A HLA typed Class I and Class II
3
Fig. 1E Supplementary Fig. S 1 B
not shown but available with author N/A N/A Supplementary Fig. S 15 and S 16 Fig. 1 (endoderm marker: E, mesoderm marker: E, ectoderm marker: A-D VI)
not shown but available with author N/A not shown but available with author
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Table 3 Reagents details. Antibodies used for immunocytochemistry/flow-cytometry Antibody
Dilution
Company Cat #; RRID
Pluripotency Marker Differentiation Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Differentiation Marker Differentiation Marker Differentiation Marker Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Differentiation Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Differentiation Marker Differentiation Marker Secondary antibody Secondary antibody Secondary antibody Secondary antibody
1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:100 1:100 1:100 1:100 1:100 1:100 1:100 1:200 1:200 1:200 1:200
Miltenyi 130-100-350; AB_2,654,226 Miltenyi 130-104-991; AB_2,653,513 Miltenyi 130-106-657; AB_2,653,536 Miltenyi 130-105-555; AB_2,653,087 Miltenyi 130-105-049; AB_2,652,991 Miltenyi 130-104-940; AB_2,653,500 Miltenyi 130-106-253; AB_2,652,977 Miltenyi 130-098-354; AB_2,655,778 Miltenyi 130-107-827; AB_2,651,758 Miltenyi 130-104-612; AB_2,661,690 Miltenyi 130-104-616; AB_2,661,695 Miltenyi 130-104-610; AB_2,661,688 Miltenyi 130-094-670; AB_10,827,599 Miltenyi 130-104-615; AB_2,661,679 Miltenyi 130-104-611; AB_2,661,677 Miltenyi 130-104-614; AB_2,661,691 Miltenyi 130-091-835; AB_871,713 Santa Cruz sc-58,669; AB_1,118,896 R&D AF1997; AB_355,097 R&D AF1759; AB_354,975 R&D AF2018; AB_355,110 eBioscience 14-8857-82; AB_11,217,879 eBioscience 14-4510-82; AB_2,572,876 BioLegend 901,301; AB_2,565,003 Biotium 20,116; AB_10,853,466 Biotium 20,014; AB_10,561,327 Biotium 20,119; AB_10,559,040 Biotium 20,012; AB_10,853,801
TRA-1-60 REA PE SSEA-1 REA PE-Vio770 SSEA-5 Mouse IgG1 VioBlue OCT3/4 Isoform A REA APC NANOG Isoform A REA APC SOX2 REA FITC Myosin Heavy Chain (MHC) REA APC CXCR4 (CD184) Mouse IgG2a PE FOXA2 REA APC REA Control S PE REA Control S PE-Vio700 REA Control S FITC Mouse IgG1 VioBlue REA control I APC REA control I FITC REA Control S APC Mouse IgG2a PE Mouse anti-α Actin Goat anti-NANOG Goat anti-OCT-3/4 Goat anti-SOX2 Mouse anti-SSEA5 Mouse anti-TUBB3 Rabbit anti-PAX6 Donkey anti-goat CF594 Donkey anti-mouse CF488 Goat anti-mouse CF594 Goat anti-rabbit CF488A
50 μM 2-mercaptoethanol and 1% penicillin-streptomycin) supplemented with 10 μM SB431542 (Miltenyi Biotec) and 1 μM LDN193189 (Selleckchem). Neural induction medium was exchanged every two days with increasing amounts (day 4–25%, day 6–50%, day 8 −75%) of neural differentiation medium (Neurobasal, 2% B-27TM Supplement minus vitamin A, 1% N-2 Supplement, 2 mM L-glutamine, 1% non-essential amino acids and 1% penicillin-streptomycin) with constant supplementation of 10 μM SB431542 and 1 μM LDN193189. At day 10 the thickened neuroectoderm was passaged by mechanical dissociation and further cultivated on GFR-Matrigel® coated plates for seven additional days in neural differentiation medium supplemented with 50 ng/ mL SHH (Peprotech), 100 ng/mL FGF-8b (Peprotech) and 20 ng/mL BDNF (Peprotech) to enrich for neural rosettes.
3.3. Direct differentiation into cardiomyocytes All four iPSC lines were differentiated successfully into beating cardiomyocytes. The StemUse101 and StemUse102 line were differentiated in 3D spheroids and the StemUse103 and StemUse105 in monolayer. For monolayer differentiation, the iPSCs were grown until 50% confluency. Subsequently, the cells were washed gently with PBS and the medium was replaced with cardiac differentiation medium (RPMI 1640 with L-glutamine supplemented with 2% B-27™ Supplement minus insulin, 50 μg/ml L-ascorbic acid 2-phosphate magnesium salt hydrate, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 1% penicillin-streptomycin) and the addition of 5 μM CHIR-99021 (LC Laboratories). After 48 h, the medium was replaced with cardiac differentiation medium with 5 μM Wnt-C59 (Cayman). After an additional 48 h, the cells were washed gently with PBS and the medium was replaced with differentiation medium with 2% B-27™ Supplement instead of B-27™ Supplement minus insulin. The latter was changed every two to three days from then on. For 3D spheroid formation 2 mL single cell suspension with 0.35 × 106 iPSCs/mL in iPSC medium with RI were placed into one well of a ULA six-well plate. After 48 h, the medium was slowly exchanged for iPSC medium, without extracting the iPSC spheroids. After an additional two days, with daily iPSC medium exchange, the cardiomyocyte differentiation was started as described above. Flow cytometry analysis of cardiomyocytes was performed when beating areas were visible. Therefore, the day of analysis was different from line to line: StemUse101 day 8, StemUse102 day 10, StemUse103 day 15 and StemUse105 day 9.
3.5. Immunocytochemistry Cells in the monolayer were fixed with 4% PFA for 10 min at RT, washed twice with PBS and incubated for 10 min with 0.2% Triton™ X100 (diluted in PBS) at RT for permeabilization. Monolayer cells in 48well plates were then incubated with the respective primary antibody (1:100) in 10% goat or donkey serum in PBS for 2 h at RT and washed afterwards twice with PBS. Fluorochrome conjugated secondary antibodies (1:200 in PBS, 45 min at RT) and 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL) were used for visualization. Images were acquired on the Keyence BZ X700E fluorescent microscope. 3.6. Flow cytometry Suspension surface staining was performed according to the manufacturer's protocol (Miltenyi Biotec). Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher), according to the manufacturer's protocol. Automatic compensation with fluorescent beads was performed according to the manufacturer's protocol (Miltenyi Biotec). Live cell discrimination was performed at the beginning of the panel setup of all different flow cytometry analyses to analyse where the living cells, dead cells and debris
3.4. Direct differentiation into neuronal cells Confluent iPSCs were differentiated from the adapted protocol of Chambers et al. by a dual SMAD inhibition into early neuroectoderm over the course of ten days (Chambers et al., 2009). In brief, iPSCs were grown until almost confluent. Differentiation was initiated by changing the medium to neural induction medium (KO DMEM, 15% KnockOut Serum Replacement, 2 mM L-glutamine, 1% non-essential amino acids, 4
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with MycoAlert® Mycoplasma Detection Kit (LT07-218) and by PCR by Eurofins Genomics Europe Applied Genomics GmbH. The sterility of cell culture supernatants was tested by the contract lab ifp (Institut für Produktqualität GmbH) according to the requirements of European Pharmacopoeia 8, 2014, section 2.6.1: test for sterility with the membrane filtration method.
populations are in the FSC/SCC plot. Data was acquired on a MACSQuant® Analyser 10 (Miltenyi Biotec) flow cytometer and analysed with the FlowLogic (Miltenyi Biotec) software. Gates were adjusted according to the respective isotype controls considering only the viable single-cell population. 3.7. Karyotyping
Supplementary materials The KaryoStat™ assay was performed by Life Technologies Corporation with iPSCs of each of the four lines in passage 17.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.scr.2019.101615.
3.8. STR analysis References STR analysis was performed by Eurofins Genomics Europe Applied Genomics GmbH. gDNA isolation was carried out from cell pellet via Chelex100 (Bio-Rad, Richmond, CA) according to the publication of Walsh et al. (Walsh et al., 2013). Genetic characteristics were determined by PCR-single-locus-technology. 16 independent PCR-systems (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, AMEL, D5S818, FGA, D19S433, vWA, TPOX and D18S51) were investigated (Thermo Fisher, AmpFlSTR® Identifiler® Plus PCR Amplification Kit). In parallel, positive and negative controls were carried out yielding correct results.
Chambers, S.M.S.M., Fasano, C.A.C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., Studer, L, ... 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat 27 (3), 275–280. Available from: http:// www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2756723&tool=pmcentrez& rendertype=abstract%5Cnhttp://www.nature.com/nbt/journal/v27/n3/abs/nbt. 1529.html. Koenig, L., Ramme, A., Faust, D., Lauster, R., Marx, U, 2018. Production of human induced pluripotent stem cell- derived cortical neurospheres in the DASbox ® mini bioreactor system. Eppend. Appl. Note. (364), 1–12. Ramme, A.P., Koenig, L., Hasenberg, T., et al., 2019. Autologous induced pluripotent stem cell-derived four-organ-chip. Futur. Sci. OA. 5 (8). Walsh, P.S., Metzger, D.A., Higuchi, R, 2013. Chelex 100 as a medium for simple extraction of DNA for PCR-Based typing from forensic material. BioTechniques 54 (3) [Internet].Available from: https://www.future-science.com/doi/10.2144/ 000114018.
3.9. Mycoplasma and bacterial testing Cell culture supernatants were tested negative for mycoplasmas
5
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Generation of four integration-free iPSC lines from related human donors ⁎
T
Anja Patricia Ramme , Daniel Faust, Leopold Koenig, Uwe Marx TissUse GmbH, Oudenarder Str. 16, 13347 Berlin, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: iPSCs Induced pluripotent stem cells Reprogramming Differentiation Human
Four integration-free iPSC lines were generated by reprogramming peripheral blood mononuclear cells with episomal vectors. All four human donors (two male and two female donors) belong to one Caucasian family within three different generations with the age between 19–82 years. Additionally, all iPSC lines are approved for commercial use by donor consent. Those iPSC lines offer the opportunity to study the influence of affiliation within one family. In future, more iPSCs lines of many more family members can be created to understand the effects of relatives with different ages on the reprogramming into iPSCs and differentiation into specific cell types.
Resource Table:
Cell line repository/bank Ethical approval
Unique stem cell lines identifier
TISSUi001-A TISSUi002-A TISSUi003-A TISSUi005-A Alternative names of stem cell li- TISSUi001-A: StemUse101, HUMIMIC101, nes SU101 TISSUi002-A: StemUse102, HUMIMIC102, SU102 TISSUi003-A: StemUse103, HUMIMIC103, SU103 TISSUi005-A: StemUse105, HUMIMIC105, SU105 Institution TissUse GmbH, Berlin, Germany Contact information of distributor [email protected], Anja Ramme Type of cell lines iPSCs Origin human Cell Source PBMCs Clonality Clonal Method of reprogramming episomes (Epi5 Kit, Thermo Fisher A15960) Multiline rationale One Caucasian family Gene modification No Type of modification N/A Associated disease 3 healthy donors, one leukemia patient Gene/locus N/A Method of modification N/A Name of transgene or resistance N/A Inducible/constitutive system N/A Date archived/stock date deposited in repository: StemUse101 06/2017 StemUse102 08/2017 StemUse103 09/2017 StemUse105 11/2017
⁎
Human Pluripotent Stem Cell Registry (hPSCreg): https://hpscreg.eu/search?q=TISSUi Ethikkomission der Ärztekammer Berlin; Eth 25/ 16
1. Resource utility There is a lack of iPSC lines with donor consent for commercial use, since many iPSC lines are only available for research. We generated four integration-free iPSC lines with donor consent for commercial use from one family to study the influence of age and gender on the reprogramming into iPSCs and differentiation into specific cell types. 2. Resource details Two male humans and two female humans from one Caucasian family (family tree in supplements Fig. S 2) with the age between 19 and 82 donated blood for iPSC generation. The integration-free reprogramming was performed by transfection with episomal vectors (Epi5 Kit, ThermoFisher A15960). The StemUse105 iPSC line was reprogrammed from a patient with leukemia, all three other lines were derived from healthy donors. Bright-field images of all four lines show colony morphology with tightly packed cells with high nucleus to cytoplasm ratio (Fig. 1A–D, I and II, scale 100 μm). The pluripotency state was characterized by positive immunofluorescence staining of NANOG, SSEA5, OCT3/4 and negative staining of α-Actin in all four iPSC lines (Fig. 1A–D, III–V, scale 100 μm). Flow cytometry staining reveals more than 95% of the cell population are positive for TRA-1-60 and negative for SSEA1, 98% are positive for SSEA5 and TRA-1-60, 92% are positive
Corresponding author. E-mail address: [email protected] (A.P. Ramme).
https://doi.org/10.1016/j.scr.2019.101615 Received 28 August 2019; Received in revised form 27 September 2019; Accepted 8 October 2019 Available online 19 October 2019 1873-5061/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Stem Cell Research 41 (2019) 101615
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Fig. 1. Characterization of the hiPSC lines TISSUi001-A (A), TISSUi002-A (B), TISSUi003-A (C) and TISSUi005-A (D). Phase contrast images of respective iPSC lines (I and II) and immunofluorescent analysis of pluripotency markers (NANOG, SSEA5, OCT3/4 and negative staining of α-Actin) (III IV). (E) Quantitative analysis by flow cytometry of the pluripotency markers (TRA-1-60+/ SSEA1, SSEA5+/ TRA-1-60+; OCT3/4+/ SOX2+ and NANOG+), definitive endoderm marker (CXCR4 and FOXA2) and mesoderm marker (MHC and cTnT).
bioinformatics assay PluriTest™ (Supplementary data Fig. S 1A). We performed monolayer-based directed differentiation of all four iPSC lines into ectoderm cells (neuronal cells), endoderm cells and
for OCT3/4 and SOX2 and 92% are positive for NANOG in all four iPSC lines (Fig. 1E and supplements Fig. S 3–S 6). The pluripotency of all four lines was further proved with the transcriptomic data based 2
Stem Cell Research 41 (2019) 101615
A.P. Ramme, et al.
Table 1 Summary of lines. iPSC line names
Abbreviation in figures
Gender
Age
Ethnicity
Genotype of locus
Disease
StemUse101 StemUse102 StemUse103 StemUse105
SU101 SU102 SU103 SU105
Male Female Female Male
52 45 19 82
Caucasian Caucasian Caucasian Caucasian
N/A N/A N/A N/A
N/A N/A N/A Leukemia
3. Materials and methods
mesoderm cells (cardiomyocytes). Neuronal differentiation was shown by positive staining for the early neuronal marker TUBB3 and the neuronal stem cell marker PAX6 in all four iPSC lines (Fig. 1A–D, VI, scale 100 μm). Definitive endoderm differentiation was performed using a commercial available kit and resulted into more than 91% CXCR4 positive cells and more than 77% FOXA2 positive cells (Fig. 1E and supplements Fig. S 7–S 10). All four iPSC lines differentiated into beating cardiomyocytes shown by the expression of myosin heavy chain (MHC) and cardiac troponin T (cTnT) (between 4% and 74% depending on the iPSC line) (Fig. 1E and supplements Fig. S 11–S 14). The KaryoStat™ assay was performed by Life Technologies Corporation and revealed no chromosomal aberrations in all four iPSC lines (Supplementary data Fig. S 1B). However, balanced translocations cannot be identified by the KaryoStat™ assay. The analysis of 15 short tandem repeats and X loci proved the identity of all four iPSC lines to the corresponding blood sample (data available with the authors). All four iPSC lines were tested negative for mycoplasma (Supplementary data Fig. S 15 and S 16) and bacterial contaminations. All data for the characterization of the lines are summarized in Tables 1–3. In the future all StemUse lines will be further differentiated in different cell types. Afterwards, those different cell types will be co-cultured in Multi-Organ-Chips (TissUse GmbH, Berlin, Germany) to study the interaction of organ models. We already showed this approach from the StemUse101 line. Four different organ models - intestine, liver, renal and neuronal models - from the StemUse101 line were differentiated and co-cultured in the four-organ-chip (HUMIMIC Chip4) and maintained for up to 14 days in a common growth factor deprived medium (Ramme et al., 2019). Additionally the StemUse101 line was used for 3D neuronal differentiation in a bioreactor system (Koenig et al., 2018).
3.1. iPSC culture Cell culture plates and components were purchased from Corning U.S. and cultures were incubated at 37 °C and 5% CO2, unless otherwise stated. The human iPSC lines HUMIMIC StemUse101, StemUse102, StemUse103 and StemUse105 (TissUse GmbH, Berlin, Germany) were derived from peripheral blood mononuclear cells. Reprogramming was performed by the service provider Phenocell SAS (Grasse, France) by transfection with episomal vectors (Epi5 Kit, Thermo Fisher A15960). The iPSCs were maintained in feeder-free conditions in StemMACS iPSBrew XF (Miltenyi Biotec) on growth factor-reduced (GFR) Matrigel® (1:100 diluted in KO/DMEM F12; Thermo Fisher) on cell culture treated dishes. The iPSCs were passaged every five to seven days using Accutase®, 4,000–6,000 cells/cm² were seeded in culture medium with 10 μM Rock Inhibitor (RI) Y-27632 (Cayman). StemMACS iPS-Brew XF medium without RI was renewed after 48 h, following a daily medium exchange.
3.2. Direct differentiation into definitive endoderm All four iPSC lines were differentiated into the definitive endoderm (DE) using the STEMdiff™ DE Kit (TeSR™-E8™ Optimized, STEMCELL Technologies), according to the manufacturer's instructions, with minor modifications. The iPSCs were split with Accutase and seeded with 33,000 cells/cm² cells on GFR Matrigel in iPSC medium supplemented with 10 μM RI and STEMdiff™ DE TeSR™-E8™ Supplement (1:20). Following medium changes were performed according to the manufacturer's instructions.
Table 2 Characterization and validation. Classification
Test
Result
Data
Morphology Phenotype
Photography Qualitative analysis (Immunocytochemistry) Quantitative analysis (Flow cytometry)
normal Positive for pluripotency markers: OCT3/4, NANOG, SOX2 and SSEA5 TRA-1-60+/ SSEA1-: 95% SSEA5+/ TRA-1-60+: 98% OCT3/4+/ SOX2+: 92% NANOG+: 92% No chromosomal aberrations were found in SU101, SU102, SU103 and SU105 when comparing against the reference dataset. DNA Profiling performed by STR analysis 16 sited tested and all matched N/A N/A Negative
Fig. 1 (A–D, I and II) Fig. 1 (A–D, III–V)
Genotype
KaryoStat™ assay
Identity
STR analysis
Mutation analysis
Sequencing Southern Blot OR WGS MycoAlert® Mycoplasma Detection Kit (LT07-218) and PCR Directed differentiation
Microbiology and virology Differentiation potential
Donor screening Genotype additional info
HIV 1 + 2 Hepatitis B, Hepatitis C Blood group genotyping HLA tissue typing
Definitive endoderm marker: CXCR4 and FOXA2 Mesoderm marker: MHC and cTnT Ectoderm marker: TUBB3 and PAX6 Negative for all four donors N/A HLA typed Class I and Class II
3
Fig. 1E Supplementary Fig. S 1 B
not shown but available with author N/A N/A Supplementary Fig. S 15 and S 16 Fig. 1 (endoderm marker: E, mesoderm marker: E, ectoderm marker: A-D VI)
not shown but available with author N/A not shown but available with author
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Table 3 Reagents details. Antibodies used for immunocytochemistry/flow-cytometry Antibody
Dilution
Company Cat #; RRID
Pluripotency Marker Differentiation Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Differentiation Marker Differentiation Marker Differentiation Marker Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Differentiation Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Pluripotency Marker Differentiation Marker Differentiation Marker Secondary antibody Secondary antibody Secondary antibody Secondary antibody
1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:11 1:100 1:100 1:100 1:100 1:100 1:100 1:100 1:200 1:200 1:200 1:200
Miltenyi 130-100-350; AB_2,654,226 Miltenyi 130-104-991; AB_2,653,513 Miltenyi 130-106-657; AB_2,653,536 Miltenyi 130-105-555; AB_2,653,087 Miltenyi 130-105-049; AB_2,652,991 Miltenyi 130-104-940; AB_2,653,500 Miltenyi 130-106-253; AB_2,652,977 Miltenyi 130-098-354; AB_2,655,778 Miltenyi 130-107-827; AB_2,651,758 Miltenyi 130-104-612; AB_2,661,690 Miltenyi 130-104-616; AB_2,661,695 Miltenyi 130-104-610; AB_2,661,688 Miltenyi 130-094-670; AB_10,827,599 Miltenyi 130-104-615; AB_2,661,679 Miltenyi 130-104-611; AB_2,661,677 Miltenyi 130-104-614; AB_2,661,691 Miltenyi 130-091-835; AB_871,713 Santa Cruz sc-58,669; AB_1,118,896 R&D AF1997; AB_355,097 R&D AF1759; AB_354,975 R&D AF2018; AB_355,110 eBioscience 14-8857-82; AB_11,217,879 eBioscience 14-4510-82; AB_2,572,876 BioLegend 901,301; AB_2,565,003 Biotium 20,116; AB_10,853,466 Biotium 20,014; AB_10,561,327 Biotium 20,119; AB_10,559,040 Biotium 20,012; AB_10,853,801
TRA-1-60 REA PE SSEA-1 REA PE-Vio770 SSEA-5 Mouse IgG1 VioBlue OCT3/4 Isoform A REA APC NANOG Isoform A REA APC SOX2 REA FITC Myosin Heavy Chain (MHC) REA APC CXCR4 (CD184) Mouse IgG2a PE FOXA2 REA APC REA Control S PE REA Control S PE-Vio700 REA Control S FITC Mouse IgG1 VioBlue REA control I APC REA control I FITC REA Control S APC Mouse IgG2a PE Mouse anti-α Actin Goat anti-NANOG Goat anti-OCT-3/4 Goat anti-SOX2 Mouse anti-SSEA5 Mouse anti-TUBB3 Rabbit anti-PAX6 Donkey anti-goat CF594 Donkey anti-mouse CF488 Goat anti-mouse CF594 Goat anti-rabbit CF488A
50 μM 2-mercaptoethanol and 1% penicillin-streptomycin) supplemented with 10 μM SB431542 (Miltenyi Biotec) and 1 μM LDN193189 (Selleckchem). Neural induction medium was exchanged every two days with increasing amounts (day 4–25%, day 6–50%, day 8 −75%) of neural differentiation medium (Neurobasal, 2% B-27TM Supplement minus vitamin A, 1% N-2 Supplement, 2 mM L-glutamine, 1% non-essential amino acids and 1% penicillin-streptomycin) with constant supplementation of 10 μM SB431542 and 1 μM LDN193189. At day 10 the thickened neuroectoderm was passaged by mechanical dissociation and further cultivated on GFR-Matrigel® coated plates for seven additional days in neural differentiation medium supplemented with 50 ng/ mL SHH (Peprotech), 100 ng/mL FGF-8b (Peprotech) and 20 ng/mL BDNF (Peprotech) to enrich for neural rosettes.
3.3. Direct differentiation into cardiomyocytes All four iPSC lines were differentiated successfully into beating cardiomyocytes. The StemUse101 and StemUse102 line were differentiated in 3D spheroids and the StemUse103 and StemUse105 in monolayer. For monolayer differentiation, the iPSCs were grown until 50% confluency. Subsequently, the cells were washed gently with PBS and the medium was replaced with cardiac differentiation medium (RPMI 1640 with L-glutamine supplemented with 2% B-27™ Supplement minus insulin, 50 μg/ml L-ascorbic acid 2-phosphate magnesium salt hydrate, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 1% penicillin-streptomycin) and the addition of 5 μM CHIR-99021 (LC Laboratories). After 48 h, the medium was replaced with cardiac differentiation medium with 5 μM Wnt-C59 (Cayman). After an additional 48 h, the cells were washed gently with PBS and the medium was replaced with differentiation medium with 2% B-27™ Supplement instead of B-27™ Supplement minus insulin. The latter was changed every two to three days from then on. For 3D spheroid formation 2 mL single cell suspension with 0.35 × 106 iPSCs/mL in iPSC medium with RI were placed into one well of a ULA six-well plate. After 48 h, the medium was slowly exchanged for iPSC medium, without extracting the iPSC spheroids. After an additional two days, with daily iPSC medium exchange, the cardiomyocyte differentiation was started as described above. Flow cytometry analysis of cardiomyocytes was performed when beating areas were visible. Therefore, the day of analysis was different from line to line: StemUse101 day 8, StemUse102 day 10, StemUse103 day 15 and StemUse105 day 9.
3.5. Immunocytochemistry Cells in the monolayer were fixed with 4% PFA for 10 min at RT, washed twice with PBS and incubated for 10 min with 0.2% Triton™ X100 (diluted in PBS) at RT for permeabilization. Monolayer cells in 48well plates were then incubated with the respective primary antibody (1:100) in 10% goat or donkey serum in PBS for 2 h at RT and washed afterwards twice with PBS. Fluorochrome conjugated secondary antibodies (1:200 in PBS, 45 min at RT) and 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL) were used for visualization. Images were acquired on the Keyence BZ X700E fluorescent microscope. 3.6. Flow cytometry Suspension surface staining was performed according to the manufacturer's protocol (Miltenyi Biotec). Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher), according to the manufacturer's protocol. Automatic compensation with fluorescent beads was performed according to the manufacturer's protocol (Miltenyi Biotec). Live cell discrimination was performed at the beginning of the panel setup of all different flow cytometry analyses to analyse where the living cells, dead cells and debris
3.4. Direct differentiation into neuronal cells Confluent iPSCs were differentiated from the adapted protocol of Chambers et al. by a dual SMAD inhibition into early neuroectoderm over the course of ten days (Chambers et al., 2009). In brief, iPSCs were grown until almost confluent. Differentiation was initiated by changing the medium to neural induction medium (KO DMEM, 15% KnockOut Serum Replacement, 2 mM L-glutamine, 1% non-essential amino acids, 4
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with MycoAlert® Mycoplasma Detection Kit (LT07-218) and by PCR by Eurofins Genomics Europe Applied Genomics GmbH. The sterility of cell culture supernatants was tested by the contract lab ifp (Institut für Produktqualität GmbH) according to the requirements of European Pharmacopoeia 8, 2014, section 2.6.1: test for sterility with the membrane filtration method.
populations are in the FSC/SCC plot. Data was acquired on a MACSQuant® Analyser 10 (Miltenyi Biotec) flow cytometer and analysed with the FlowLogic (Miltenyi Biotec) software. Gates were adjusted according to the respective isotype controls considering only the viable single-cell population. 3.7. Karyotyping
Supplementary materials The KaryoStat™ assay was performed by Life Technologies Corporation with iPSCs of each of the four lines in passage 17.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.scr.2019.101615.
3.8. STR analysis References STR analysis was performed by Eurofins Genomics Europe Applied Genomics GmbH. gDNA isolation was carried out from cell pellet via Chelex100 (Bio-Rad, Richmond, CA) according to the publication of Walsh et al. (Walsh et al., 2013). Genetic characteristics were determined by PCR-single-locus-technology. 16 independent PCR-systems (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, AMEL, D5S818, FGA, D19S433, vWA, TPOX and D18S51) were investigated (Thermo Fisher, AmpFlSTR® Identifiler® Plus PCR Amplification Kit). In parallel, positive and negative controls were carried out yielding correct results.
Chambers, S.M.S.M., Fasano, C.A.C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., Studer, L, ... 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat 27 (3), 275–280. Available from: http:// www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2756723&tool=pmcentrez& rendertype=abstract%5Cnhttp://www.nature.com/nbt/journal/v27/n3/abs/nbt. 1529.html. Koenig, L., Ramme, A., Faust, D., Lauster, R., Marx, U, 2018. Production of human induced pluripotent stem cell- derived cortical neurospheres in the DASbox ® mini bioreactor system. Eppend. Appl. Note. (364), 1–12. Ramme, A.P., Koenig, L., Hasenberg, T., et al., 2019. Autologous induced pluripotent stem cell-derived four-organ-chip. Futur. Sci. OA. 5 (8). Walsh, P.S., Metzger, D.A., Higuchi, R, 2013. Chelex 100 as a medium for simple extraction of DNA for PCR-Based typing from forensic material. BioTechniques 54 (3) [Internet].Available from: https://www.future-science.com/doi/10.2144/ 000114018.
3.9. Mycoplasma and bacterial testing Cell culture supernatants were tested negative for mycoplasmas
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