All studies were approved by the Institutional Review Board and Institutional Animal Care and Use Committee.
GMP-grade HPCs were originally harvested from patients for stem cell therapy. Specifically, HPCs were harvested after mobilization by injection with granulocyte colony-stimulating factor for 5 days in the Human Cellular Therapy Laboratory at the Mayo Clinic. Those cell products are routinely discarded as clinical wastes when patients have died before the initiation of stem cell therapy. We received de-identified clinical-waste HPCs from the Human Cellular Therapy Laboratory for iPSC derivation. The use of clinical-waste HPCs for iPSC generation was approved by the Institutional Review Board, including the Biospecimens Subcommittee. For mononuclear myeloid cell derivation, 10 ml of whole blood was purchased and used to isolate PBMCs, as reported previously [33, 34].
Lentiviral vector production
Pluripotency-associated factor-expressing lentiviral vectors, pSIN-OCT4, pSIN-SOX2, pSIN-KLF4, and pSIN-cMYC were described previously . These vectors were produced by transient transfection of 293T cells. Vector titers were determined with immunostaining .
HPCs and PBMCs were cultured overnight in StemSpan H3000 serum-free medium (StemCell Technologies, Vancouver, Canada), which contains only human-derived or recombinant human proteins, supplemented with StemSpan CC100 cytokine cocktail (StemCell Technologies). Cells (105 cells/500 μl of medium in each well) in uncoated wells of a 24-well plate were then transduced with four stemness factor-expressing lentiviral vectors overnight. One third of the culture supernatants were carefully removed and replaced daily with H3000 growth medium supplemented with CC100 cytokine cocktail. Three days after vector infection, cells in a well of a 24-well plate were transferred to a Matrigel (BD Biosciences, Bedford, MA)-coated well of a 6-well plate. Starting 5 days after vector infection, cells were maintained in HEScGRO medium (100 ml; Millipore, Billerica, MA) supplemented with mTeSR-1 maintenance media (25 ml; StemCell Technologies) . 7 to 10 days after vector infection, the reprogrammed cells began to form colonies with iPS morphology. At 2 to 3 weeks after vector infection, cultures were treated with Cell Dissociation Buffer (Invitrogen, Grand Island, NY) for 5 to 10 minutes to help lift clones, and individual iPSC-like clones were carefully picked up with a P200 pipette, and placed into Matrigel-coated wells in a 96-well plate. To prevent spontaneous differentiation, the iPSC culture medium was replaced daily, and differentiated cells in the cultures were manually removed with a pipette tip. As the clones grew, cultures were expanded into larger culture plates for further characterization. Clones were preserved by using Xeno-FREEze Human Embryonic Stem Cell Freezing Medium (Millipore). A verified iPSC clone, HCF1, from primary human fibroblast (HCF) cells, was described previously . Primary human keratinocytes and keratinocyte-derived iPSC clones were also used as controls .
Immunostaining and alkaline phosphatase staining
For immunostaining of iPSC, cells were fixed for 20 minutes at room temperature in 4% paraformaldehyde solution in PBS, washed several times in PBS, and blocked for 30 minutes in PBS with 5% fetal bovine serum. Cells were then stained with primary antibodies overnight at 4°C, rinsed with PBS, and incubated with secondary antibodies for 1 hour at RT. For immunostaining of differentiated cells, cells at different stages of differentiation were fixed and stained with primary and secondary antibodies. Primary antibodies used for characterization of iPSC and iPSC-derived cells were SSEA-4 and TRA-1-60 (Millipore, #SCR001), OCT4 (Cell Signaling Technology, #2750, Danvers, MA), NANOG (Abcam, #ab21624, Cambridge, MA), mouse anti-SOX17 (R&D Systems, #MAB1924, Minneapolis, MN), rabbit anti-HNF3 beta/FOXA2 (Millipore, #07-633), rabbit anti-PDX1 (Santa Cruz Biotechnology, #sc-25403, Santa Cruz, CA) and mouse anti-insulin (Sigma, #I2018, St. Louis, MO). Texas Red-conjugated donkey-anti-rabbit IgG (Jackson ImmunoResearch Laboratories, #711-075-152, West Grove, PA), Texas Red-conjugated donkey-anti-mouse IgG (Jackson ImmunoResearch Laboratories, #715-075-151), FITC-conjugated donkey-anti-rabbit IgG (Jackson ImmunoResearch Laboratories, #711-095-152), and FITC-conjugated donkey-anti-mouse IgG (Jackson ImmunoResearch Laboratories, #715-095-151) were used as secondary antibodies. DAPI was used for counterstaining. Stained cells were analyzed by using a confocal laser-scanning microscope (Zeiss, LSM 510 confocal scanning laser system).
For spontaneous differentiation, iPSC clones were dissociated by using collagenase IV for 30 minutes and plated on low-adhesion plates in basal HEScGRO medium without bFGF. Embryoid bodies (EBs) were cultured as a suspension for 7 to 10 days and adherent in DMEM with 20% FBS for additional 7 to 10 days. For immunofluorescence analysis, cells were fixed with 4% PFA for 20 minutes at RT. Immunostaining was performed as described earlier. Primary antibodies against FOXA2 for endoderm, beta-III tubulin (Abcam, #41489) for ectoderm, and CD31 (Santa Cruz Biotechnology, #SC1506) for mesoderm were used, whereas Texas Red-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, #711-075-152), and FITC-conjugated donkey anti-chicken IgG (Jackson ImmunoResearch Laboratories, #703-095-155) served as secondary antibodies.
In vivo differentiation of derived iPS cells
SCID-beige mice were anesthetized, and the kidney was externalized for iPS transplantation under the kidney capsule. A small incision was made in the kidney capsule, and a blunt needle used to create a pocket under the kidney capsule. After iPSC injection into the pocket, the kidney was placed back into the abdomen, and the incision closed with a Vicryl suture. Mice were maintained for 4 weeks and killed for harvesting normal and iPS-transplanted kidneys. OTC-embedded frozen tissues were cryo-sectioned for H&E staining.
Differentiation of derived iPS cells into insulin-producing cells iPSCs were differentiated into insulin-producing cells, as reported previously with minor modifications . At the first step of differentiation, human iPSC clones were treated with 25 ng/ml Wnt3a (R&D Systems) and 100 ng/ml activin A (Peprotech, Rocky Hill, NJ) in advanced RPMI (Invitrogen) with Pen/Strep for 1 day, followed by treatment with 100 ng/ml activin A in advanced RPMI supplemented with 0.2% fetal calf serum (FBS) (Invitrogen) for 2 days. At step two, cells were cultured in high-glucose DMEM (Invitrogen), supplemented with 20% (vol/vol) advanced RPMI medium containing 50 ng/ml FGF10 (R&D systems), 0.25 μmol/L KAAD-cyclopamine (CYC), and 2% FBS for 2 days. Cells were then treated with 50 ng/ml FGF10, 0.25 μmol/L CYC, and 2 μmol/L all-trans retinoic acid (RA) (Sigma) in high-glucose DMEM (Invitrogen) supplemented with 20% advanced RPMI, Pen/Strep, 1 × B27 supplement (Invitrogen) for 4 days at step three. Cells were then cultured in the presence of 50 ng/mL FGF10, 300 nmol/L ILV (Axxora, San Diego, CA), and 55 nmol/L GLP-1 (Sigma) in DMEM (high glucose) supplemented with 20% advanced RPMI and 1 × B27 for 4 days at step four. In step five, differentiation medium included 10 μmol/L DAPT (Sigma) and 55 nmol/L GLP-1 in DMEM (high glucose) with 20% advanced RPMI and 1 × B27, and the culture lasted for 6 days.
Finally, in step six, cells were cultured in the presence of 50 ng/ml hepatocyte growth factor (HGF) (R&D systems), 50 ng/ml insulin-like growth factor 1 (IGF-1) (R&D Systems), and 55 nmol/L GLP-1 in CMRL-1066 medium (Invitrogen) with 1 × B27 for 8 days. All differentiation experiments were performed in triplicate, and repeated at least twice.
Total RNA was isolated by using TRIzol (Invitrogen) and further purified by using RNeasy Plus spin columns (QIAGEN, Valencia, CA). Turbo DNA-free DNase (Ambion, Austin, TX) was used to digest all genomic DNA that could lead to false-positive gene-expression results. The RNA quantity and purity was measured with a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE), and the RNA integrity was determined by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Microarray analysis was performed by using the Affymetrix HG-U133 Plus2 GeneChip Array platform (Affymetrix, Santa Clara, CA). Data were preprocessed by using standard in-house MicroArray Pre-Processing workflow, and hierarchical clustering was performed by Pearson Dissimilarity. To compare the transcriptome of blood-derived iPSCs, the data set of epidermal keratinocytes (HK, SW3, SW4, and SW8), two keratinocyte-derived iPSC clones (SW3 b and SW4 N1) and human fibroblast (FB)-derived iPSC clone HCF1  were used. A t test was performed to analyze the significance of the changes (P < 0.05) in the normalized gene-expression levels between HK and iPSC clones, or between blood-derived iPSC clones and HK- and FB-derived iPSC clones. Heatmap Builder software (kindly provided by Dr. Euan Ashley, Stanford School of Medicine) was used to generate the heatmap for the transcriptome data set. The registered GEO transcriptome database (GSM551202, human ES H9 cells; GSM452255, freshly isolated PBMC; GSM178554, mobilized HPCs) was used to analyze the similarities between blood-derived iPSCs and human ES cells or non-reprogrammed PBMCs and HPCs. Microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE33536 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33536).