Defined Xenofree Reprogramming of Cord Blood Progenitors to Induced Pluripotent and Blastomere-Like Stem Cells

Summary

This protocol outlines chemically-defined methods for xenofree (XF), feeder-free (FF), episomal reprogramming of human cord blood (CB) myeloid progenitors to conventional human induced pluripotent stem cells (hiPSC). These XF/FF CB-hiPSC (XF-hiPSC) can be efficiently reverted to blastomere-like Tankyrase/PARP1 Inhibitor-Regulated Naive stem cells (TIRN-SC) possessing augmented differentiation and interspecies chimera potential.

Abstract

Human cord blood (CB) myeloid progenitor reprogramming to a high-fidelity human induced pluripotent stem cell (hiPSC) state can be achieved using non-integrating episomal vectors and stromal signals. These conventional, primed CB-hiPSC lines can subsequently be chemically reverted with high efficiencies to a blastomere-like Tankyrase/PARP Inhibitor-Regulated Naive Stem Cell (TIRN-SC) state with functional totipotency. PARP-regulated TIRN-SCs are human stem cells with high epigenetic plasticity, stable epigenomic imprints, and have greater differentiation potency than conventional, lineage-primed hiPSCs. Here, optimized XF/FF methods are outlined for efficient mesenchymal stroma-activated episomal reprogramming of CD34+ CD33+ CB myeloid progenitors into conventional XF/FF hiPSC. TIRN reversion reproducibly potentiated XF/FF conventional hiPSC to adopt transcriptional, epigenetic, and functional features of cleavage-stage human embryo cells with decreased lineage-primed gene expression. We validated that TIRN-reverted CB-derived XF-hiPSC displayed marked improvement in directed multi-lineage differentiation (including hematovascular lineages) across a broad repertoire of genetically independent backgrounds. These methods serve as a first step for generating cGMP-compliant TIRN-SC lines for clinical-grade HLA-defined 'Universal' donor TIRN-SC (UTIRN-SC) banks. The derivation of UTIRN-SC lines with improved differentiation versatility from CD34+ CD33+ CB progenitors could have a high impact on regenerative medicine. For example, UTIRN-SCs could generate tissue banks of HLA-defined, cryo-preserved cardiac, vascular, and neural donor progenitors for comprehensive multi-lineage "off-the-shelf" cellular therapies.

Introduction

Although human induced pluripotent stem cells (hiPSCs) offer promising opportunities for personalized cellular therapies, their clinical application faces major challenges, including tumorigenicity, immunogenicity, and phenotypic and functional heterogeneity¹, which impact their safety, efficacy, and cost-effectiveness. Furthermore, lineage-committed progenitors derived from conventional, primed hiPSC lines are generated with highly variable interline differentiation efficiencies², and exhibit limited in vivo engraftment and functionality. However, such limitations can be overcome using optimized reprogramming methods³ or alternative stem cell states^4,5.

Although nonviral episomal reprogramming efficiencies of skin or blood somatic cells into hiPSC are low (~0.01%-2%)⁶, highly optimized four-factor reprogramming of CB progenitors can be attained and can reach up to 50% bulk efficiencies in mesenchymal stromal stem-progenitor (MSC)-activated co-culture conditions⁷. In this system, cellular reprogramming of CB myeloid progenitors was accelerated by soluble and contact-dependent stromal signals. Stroma-primed (SP) episomal CB-derived hiPSC lines differentiated into vascular progenitors (VP) with higher efficiencies than conventional fibroblast-derived hiPSC^3,5. SP-CB-iPSC-derived VP exhibited more authentic embryonic VP transcriptional identity, reduced senescence, and sensitivity to DNA damage^3,4. SP-CB-iPSC-derived VP also demonstrated more robust in vivo engraftment than conventional hiPSC-derived VP following systemic or direct transplantation into the vitreous of retinal ischemia-reperfusion-injured adult immunodeficient mice^3,4.

Zimmerlin et al. established a two-step chemical reprogramming system ('LIF-5i -> LIF-3i') that transitions primed, conventional human pluripotent stem cells to a TIRN-SC state with improved multi-lineage differentiation potential⁵. Differentiated TIRN-SC-derived vascular progenitors exhibited higher functionality, greater genomic stability, and superior in vivo engraftment, as shown by their improved capacity for migrating to and re-vascularizing the deep neural layers of the ischemic retina⁴. TIRN-SC underwent proteogenomic reprogramming to acquire a functional hybrid blastomere-like state with high contribution in the interspecific chimera assay⁸. TIRN-SC maintained DNMT1 expression and were protected against erosion at CpG-methylated genomic imprinted regions. Interestingly, SP-CB-hiPSC were reverted into TIRN-SC more efficiently than hiPSC derived via alternative, less efficient methods^5,9.

Thus, TIRN-SCs are an alternative stem cell state with improved multi-lineage differentiation capacity and efficient chimera contribution. Further optimization of
TIRN-SC reprogramming in defined, cGMP-compliant culture conditions will facilitate the generation of a broad array of functional, engraftable cell types and tissues for therapies. To address the need for cost-effective therapeutic progenitors, these methods can be applied to produce cGMP-grade banks of 'Universal Donor' TIRN-SC (UTIRN-SC) using HLA-defined CD34⁺ hematopoietic CB or peripheral blood progenitors. HLA-defined UTIRN-SC-derived progenitors could serve the needs of a larger number of patients requiring immediate, multi-lineage regeneration of complex tissues.

The paradigms developed for clinical bone marrow transplantation (BMT) may guide the development of UTIRN-SC banks from partially HLA-matched, or HLA-haplo-identical hematopoietic stem cells from national donor registries. A predictive, global computational analysis of the combined top 10 haplotypes from 18 countries, including the USA, would provide a mean patient coverage of 68.4% from such hiPSC 'haplobanks'¹⁰. A defined UTIRN-SC bank could not only provide lineage-committed progenitors, but also support therapeutic TIRN-SC applications of interspecific blastocyst complementation systems within domestic animals (e.g., pigs) for whole organ generation^11,12, including generation of tolerance-inducing hematopoietic progenitors for reducing graft rejection in populations with broad genetic diversity (e.g., USA).

This modified reprogramming protocol for generating TIRN-competent XF/FF hiPSC lines is a first step for developing future cGMP-compliant UTIRN-SC banks. This protocol outlines stepwise stroma-primed reprogramming and functional validation of human CB myeloid progenitors into conventional XF-hiPSC lines. These XF-hiPSC demonstrated facile reversion into TIRN-SC, with efficiency similar to non-XF methods⁷ and attained unrestricted functional pluripotency, including improved capacity to produce well-formed lineage bias-reduced teratomas (relative to isogenic primed XF-hiPSC)^4,5,9. We also outline protocols that validate TIRN-reverted XF-hiPSC for their improved and robust direct differentiation to hematovascular lineages.

Protocol

All reprogramming experiments conformed to the guidelines published by the National Academy of Sciences, and the International Society of Stem Cell Research (ISSCR). For hiPSC generation, donor cell collection protocols and donor-informed consent were approved by the Johns Hopkins University (JHU) Institutional Review Board (IRB) oversight. All experiments were conducted under purview of the JHU Institutional Stem Cell Research Oversight (ISCRO) and conform to Institutional standards regarding informed consent and provenance evaluation. Additionally, all animal use and surgical procedures were performed in accordance with protocols approved by the JHU Institute of Animal Care and Use Committee (IACUC). All experiments involving animal procedures (e.g., subcutaneous/intra-muscular injections of NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NOG SCID) mice (Jackson Labs, Bar Harbor, ME) for teratoma analysis and blastocyst transfer into ICR pseudopregnant mice) were reviewed and approved by the JHU IACUC. These reviews included considerations for ethical sacrifice, humane housing, and appropriate measures to minimize animal discomfort.

1. Feeder-free xenofree hiPSC episomal reprogramming

1. Donor hematopoietic progenitor cell preparation
   1. Use cryopreserved CD34⁺ hematopoietic stem/progenitor cells (e.g., 1x10⁶ neonatal CB cells) immediately upon delivery or store cryovials in a liquid nitrogen storage tank. Make sure cells have been continuously maintained in a frozen state during transport and handling.
      NOTE: Alternatively, non-cryopreserved donor cells can also be utilized immediately following collection and CD34 enrichment using approved protocols.
   2. Before thawing cells in the water bath, gently open the sterilized cryovial within a biosafety cabinet to release any trapped gas inside the vial and then tightly close the vial.
   3. Place the vial in a 37 °C water bath for 1 to 2 min. Gently swirl the vial until almost no ice remains. Remove the vial from the water bath and thoroughly sterilize it (i.e., 70% ethanol spray).
      NOTE: All steps require proper aseptic technique and are performed within a sterile biological safety hood cabinet.
   4. Gently and slowly transfer the cells into an empty 50 mL sterile conical tube. Wash the vial with 1 mL of cold xenofree hematopoietic progenitor (XF-HP) expansion medium (Table 1) and gently combine (dropwise along the edges of the tube) with the cells to dilute the DMSO cryoprotectant.
   5. Leave the tube standing up in a rack for 30 s and slowly (dropwise) add 2 mL of cold XF-HP medium. Gently swirl cell suspension while adding the medium. Repeat this step twice by adding sequentially 4, 8 and 4mL of cold XF-HP medium (up to 20 mL total volume). Centrifuge the diluted cells at 100 x g for 10 min at 4 °C.
      NOTE: Do not pipet cells up and down in the cryoprotectant solution and during the dilution steps. Add XP-HP medium dropwise and slowly until the DMSO is diluted at least 10-fold. Use cold medium as DMSO dilution results in an exothermic reaction.
   6. Aspirate cell-free supernatant in a biosafety cabinet. Gently dislodge the cell pellet. Resuspend in 2 mL of XF-HP expansion medium for cell counting using a hemocytometer or an automatic cell counter.
   7.  Transfer the thawed cells onto ultra-low attachment cell culture multi-well plates (e.g. transfer 2 mL of cell/XF-HP medium mixture into 1 well of a 6-well plate).
   8. Tightly wrap the plate in Saran wrap for mimicking hypoxic conditions. Transfer the plate in an incubator (37 °C , 5% CO₂, humid atmosphere). Alternatively, place the unwrapped plate in an incubator with O₂ control (5% O_2, 5% CO₂, humid atmosphere).
   9. Culture the hematopoietic progenitors for 3 days in XF-HP medium.
2. Mesenchymal stem-progenitor cell (MSC) preparation
   1. Thaw an aliquot (0.75 - 1x10⁶ cells) of low passage (p2 to p3) human MSC by incubating a cryopreserved aliquot in a 37 °C water bath for 1 to 2 min. Gently swirl the vial until almost no ice remains. Remove the vial from the water bath and thoroughly sterilize (i.e., 70% ethanol).
      NOTE: Low passage (p2) human MSC can be purchased or prepared from human bone marrow using established protocols and cryopreserved. MSC can be thawed about 7-10 days in advance and irradiated/plated as a feeder layer the day prior to nucleofection  (Figure 1).
   2. Gently and slowly (dropwise along the edges of the tube) transfer the cells into a 15 mL conical tube containing cold xenofree serum-free mesenchymal stem cell (XF-MSC) medium (Table 1) within a biosafety cabinet to dilute the DMSO cryoprotectant at least 10-fold (e.g., transfer 1 mL cryopreserved cell aliquot into 9 mL XF-MSC medium).
   3. Use 1 or 2 mL of cold XF-MSC medium to rinse the tube and combine into the conical tube. Centrifuge the diluted cells at 100 x g for 5 min at 4 °C.
   4. Aspirate cell-free supernatant in a biosafety cabinet. Gently resuspend the cell pellet in 1 mL of XF-MSC medium for cell counting using a hemocytometer or an automatic cell counter.
   5.  Transfer the thawed cells onto tissue-treated cell culture multi-well plates (e.g. 5,000-6,000 cells per cm² or about 50,000 cells in 1 well of a 6-well plate). Place plates in an incubator (37 °C, 5% CO₂, humid atmosphere). Refresh XF-MSC medium every 2-3 days within a biosafety cabinet.
   6. The day prior to nucleofection, aspirate cell-free supernatant in a biosafety cabinet. Wash MSC once with PBS (2 mL/well). Aspirate PBS. Add 1 mL/well of cell dissociation reagent (e.g., 1X Trypsin-EDTA solution). Incubate for 5 min at 37 °C in a CO₂ incubator.
   7. Neutralize the dissociation reagent by adding 1 mL of XF-MSC medium (at least 2-fold dilution) and gently triturate with a pipette into a single cell suspension. Collect the cell suspension into a 15 mL conical tube. Centrifuge at 100 x g for 5 min.  
   8. Aspirate the supernatant. Resuspend the cell pellet in 1 mL of XF-MSC medium for cell counting using a hemocytometer or an automatic cell counter.
   9. Irradiate (2,000 rad) mesenchymal progenitors under 5 passages using a g- or X-ray- irradiator and plate onto Arg-Gly-Asp (RGD) peptide-coated synthetic surface 6-well plates (2x10⁵ cells per well) in XF-MSC medium. Keep plates overnight in an incubator (37 °C, 5% CO₂, humid atmosphere).
3. Xenofree episomal reprogramming
   1. Collect 3-day-old CB HP cultures in XF-HP expansion medium into a 15 mL conical tube in a biosafety cabinet. Centrifuge at 200 x g for 5 min.
   2. Aspirate cell-free supernatant in a biosafety cabinet.  Gently resuspend the cell pellet in 1 mL of XF-HP expansion medium for cell counting using a hemocytometer or an automatic cell counter.
   3. Combine 1x10⁶ expanded HP cells with 100 µL of human CD34⁺ cell nucleofection solution (human CD34⁺ cell nucleofector kit). Add 6 µg of purified concentrated episome (<5 µL) and carefully transfer into a nucleofector cuvette using the plastic transfer pipet of the nucleofection kit. Avoid bubbles. Place the cuvette in the nucleofector device and use program U-008.
      NOTE: Prepare stocks of the episomal EBNA-based pCEP4 vector pEP4 EO2S EM2K (OCT4, SOX2, MYC, KLF4) by routine transformation in TOP10 E. coli and purify using plasmid Maxi-prep kits. The day of nucleofection, clean up and concentrate the plasmid using a PCR purification kit ⁷.
   4. Immediately following nucleofection, add 500 µL of pre-warmed XF-HP expansion medium into the cuvette and transfer the cells in 1 mL of XF-HP medium onto 1 well of ultra-low attachment cell culture 6-well plate using a sterile transfer pipet.
      NOTE: Nucleofected cells are very fragile and will not survive the shear stress from regular 1 mL pipet tips. Transferring onto ultra-low attachment culture plates is a step that promotes post-nucleofection cell recovery before final attachment onto RGD-coated plates.
   5. Tightly wrap the plate in plastic wrap for mimicking hypoxic conditions. Place the plate in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere). Alternatively, place the unwrapped plate in an incubator with O₂ control (5% O_2, 5% CO₂, humid atmosphere). Incubate for 4 to 6 hours.
      NOTE: This step will allow nucleofected HP cells to recover before transferring onto MSC monolayers.
   6. Collect the cells into a 15 mL conical tube in a biosafety cabinet. Centrifuge at 200 x g for 5 min.
   7. Aspirate the supernatant within a biosafety cabinet. Resuspend the cell pellet in 1 mL of XF-HP expansion medium. Plate nucleofected HP cells onto irradiated MSC at a density of about 17,500 cells per cm² in RGD-coated plates in XF-HP expansion medium (2 mL/well) supplemented with ROCK inhibitor Y-27632 (final concentration 10 µM). Place the plate in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere).
   8. Two days after nucleofection, add 2 mL (equal volume) of E8 medium in each well within a biosafety cabinet.
   9. Replace half of the volume of culture medium every other day with E8 medium. Collect floating cells into a 15 mL conical tube, centrifuge at 200 x g for 5 min, and add the recovered cells back to the culture.
   10. On day 21, prepare vitronectin-coated tissue culture-treated 6-well plates. Add 1mL of diluted vitronectin solution (e.g., 40 mL/mL for Vitronectin-XF) to 1 well of a 6-well plate and incubate for 1 hour at room temperature.
   11. Manually pick individual XF-hiPSC colonies using an inverted microscope within a biosafety cabinet. Triturate with pipet and transfer single colonies onto vitronectin-coated plates in E8 medium supplemented with ROCK inhibitor Y-27632 (final concentration 5 µM). Place the plate in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere).
   12. The next day, carefully replace the medium with E8 medium without ROCK inhibitor supplementation. Change E8 medium everyday thereafter (2 mL/well) in a biosafety cabinet.

2. Routine hiPSC culture and maintenance

1. When the colonies become larger 5 to 6 days later (i.e., 0.5 to 1 mm diameter or about 80% confluence), aspirate the medium in a biosafety cabinet.
2. Add 2 mL/well PBS to wash the cells. Aspirate the PBS. Add 1 mL EDTA-based cell dissociation solution. Incubate for 5 min in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere). Check that the cells are loosely attached (e.g., shiny round cells, but still attached to the plate) using a microscope.
3. Gently aspirate the dissociation solution within a biosafety cabinet without disrupting the cells.
4. Collect the cells in 1-2 mL of E8 medium supplemented with ROCK inhibitor Y-27632 (final concentration 5 µM) with non-vigorous pipetting to maintain cell clumps. Transfer cell clumps onto vitronectin-coated (see section 1.3.11) 6-well cell culture-treated plates (equivalent of 10 colonies per well or 1:10 split). Place the plate in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere).
5. Continue sub-culturing using EDTA-based cell dissociation solution every 5-7 days.
   NOTE: Additional sub-cloning or manual colony picking is not usually required for SP-CB-hiPSC reprogramming, but if hiPSC clones contain colonies with abnormal morphology (e.g., spontaneous differentiation such as fibroblasts or embryoid body formation, emergence of abnormal non-flat colonies), manually select colonies using a micro-pipet and a microscope within a biosafety cabinet or discard aberrant clones. Non-efficient, non-stable reprogramming of hiPSC is not compatible with TIRN-SC reversion.

3. Phenotypic validation of XF-hiPSC clones

1. Expand XF-hiPSC clones for at least 5-7 passages for initial validation (Figure 2) as described in section 2, prior to long-term cryopreservation and application in functional studies. Record the number of passages on each cryopreserved vial.
   NOTE: Initial characterization of low passage XF-hiPSC clones can be confirmed by multiple assays such as akaline phosphatase activity, and TRA-1 antigen or NANOG immunodetection.
2. Perform XF-hiPSC validation and enumeration with alkaline phosphatase staining (Figure 2A,B).
   NOTE: Reprogramming efficiencies in bulk cultures can be determined at P0 3-5 weeks post-episomal nucleofections by enumerating the number of fully reprogrammed colonies per single input cells plated on day 3^7,13.
   1. Aspirate culture medium in a biosafety cabinet.
   2. Wash cells with PBS (2 mL per well). Aspirate PBS. Fix cells by adding 2% paraformaldehyde/PBS solution. Incubate for 10 min at room temperature.
   3. Remove and dispose of paraformaldehyde/PBS solution according to chemical waste safety guidelines.
   4.  Wash cells with PBS (2 mL per well). Aspirate PBS. Add 1-2 mL of alkaline phosphatase substrate and incubate for 10 to 15 min at room temperature with slow agitation.
   5. Wash 3 times with PBS to stop the reaction. Photomicrograph and enumerate.
      NOTE: Avoid excessive incubation time or temperature as it can result in false positive staining.
3. Perform XF-hiPSC staining validation and enumeration with TRA-1 antigen immunostains (Figure 2C).
   ​NOTE: For episomal reprogramming, detection of transgenic expression and EBNA1 vector backbone during initial passages should be performed using published protocols⁷. Routine validation includes karyotyping by G-banding (Figure 2D) or whole-genome array-based coverage assays. Basic functional pluripotency can also be assayed using teratoma differentiation protocols (Figure 2E) or quantitative multi-gene expression scorecards of non-directed embryoid body differentiations¹⁴.
   1. In a biosafety cabinet, immunostain replicate wells for TRA-1-60 or TRA-1-81 antigens for 30 min by adding fluorochrome-conjugated live-stain antibodies to E8 medium at the appropriate concentration.
   2. Aspirate the culture medium. Wash twice with E8 medium (2 mL per well).
   3. Photomicrograph and enumerate in E8 medium or Phenol Red-free medium.
      NOTE: Alternatively, fix cells using 2% paraformaldehyde/PBS solution for 10 min at room temperature and immunostain for TRA-1 antigens using standard antibodies. 
4. Validate XF-hiPSC immunophenotype by flow cytometry.
   1. In a biosafety cabinet, aspirate culture medium.
   2. Wash cells in PBS (2 mL per well). Aspirate PBS. Add enzymatic dissociation reagent (e.g., Accutase, 1mL per well). Incubate the plate for 5 min at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere).
   3. Collect cells by adding an equal volume of culture medium and pass through a cell strainer (40-70 µm filter). Centrifuge cells at 200 x g for 5 min.
   4. Resuspend cell pellet in PBS with 5% FBS (1 mL per well-equivalent). Count the number of cells using a hemocytometer or an automatic cell counter. Distribute about 100,000-200,000 cells in round bottom tubes per FACS assay (keeping volumes at about 100 µL per assay).
   5. For extracellular stainings, after an optional blocking step using neat mouse serum (add 5 µL per assay, 5 min at room temperature), incubate with fluorochrome-conjugated antibodies (e.g., TRA-1-81 PE, SSEA4 APC) according to the manufacturer’s instructions. Wash with 3 mL PBS. Centrifuge for 5 min at 300 x g. Resuspend in FACS buffer (e.g., PBS with 5% FBS) and acquire using a FACS instrument.
      NOTE: always use appropriate isotype controls and stain parallel samples to validate the specificity of the immunodetected proteins.
   6. For intracellular stainings (e.g., NANOG-PE, OCT3/4-PE, SOX2-PE), follow manufacturer’s instructions for fixation/permeabilization steps. Incubate with fluorochrome-conjugated antibodies or matching isotype controls. Acquire using a FACS instrument.

4. Cryopreservation and thawing of XF-hiPSC clones

1. Expand XF-hiPSC clones for at least 5-7 passages to perform initial validation (Figure 2) before long-term cryopreservation and application in functional studies. Record the number of passages on each cryopreserved vial.
   1. Aspirate culture medium in a biosafety cabinet.
   2. Wash cells with PBS (2 mL per well). Aspirate PBS.  Add EDTA-based cell detachment solution (1 mL per well) to dissociate XF-hiPSC colonies into cell clumps. Incubate the plate for 5 min in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere). Check that the cells are loosely attached (e.g., shiny round cells) using a microscope.
   3. Gently aspirate dissociation solution without disrupting the cell monolayer colonies and collect the cells in 1-2 mL E8 medium with non-vigorous pipetting to maintain cell clumps (10-50 cells per clump). Collect XF-hiPSC into a sterile 15 mL conical tube. Centrifuge cells at 200 x g for 5 min.
   4. Aspirate the supernatant in a biosafety cabinet. Resuspend cell pellet in freezing solution (Table 2, ~ 1 well equivalent per mL).
   5. Transfer cells into long-term storage cryogenic tubes. Place the tubes into a slow freezing container and allow the samples to freeze overnight in a -80 °C freezer. The next day, place the cryovials into a liquid nitrogen freezer for long term storage.
2. For thawing, prepare a plate by pre-coating with vitronectin (see section 1.3.11).
   1. Place the frozen vial into a 37 °C water bath and thaw the cells for ~1-2 min. Sterilize (i.e., ethanol spray).
   2.  Slowly transfer XF-hiPSC into a sterile 15 mL conical tube that contains 9 mL of thawing medium (Table 2) to slowly dilute cells 10-fold within a sterile biological safety hood cabinet.
      NOTE: Do not pipet vigorously.
   3. Centrifuge at 200 x g for 5 min.
   4. Aspirate  the cell-free supernatant in a biosafety cabinet. Resuspend the cell pellet in E8 medium (1-2 mL) supplemented with ROCK inhibitor (final concentration 5 mM).
      Note: Exclusion of Y-27632 will result in poor post-thawing recovery efficiencies.
   5. Transfer the thawed cells onto 1 or 2 vitronectin-coated wells of a 6-well plate. The next day, change medium with regular E8 medium without ROCK inhibitor for cell expansion.

5. Xenofree hematovascular differentiation of XF-hiPSC

1. Differentiate XF-hiPSC using our optimized xenofree hemato-vascular differentiation protocol (Figure 3)⁴.
   ​NOTE: This protocol is modified from¹⁵.
   1. Prepare XF-hiPSC at ~40-50 % confluence (1-3 days after passaging).
   2. Aspirate culture medium and add 2 mL mesodermal determination medium (Table2) in a biosafety cabinet.
   3. After 2 days, aspirate the medium and add 2 mL/well of vascular differentiation medium (Table2) in a biosafety cabinet. If a lot of dead or floating cells are visible at this step, wash once with 2 mL PBS.
   4. Replace vascular differentiation medium every other day in a biosafety cabinet up to day 11.
      NOTE: If medium turns yellow due to high cell density and low pH, add 1 mL of fresh medium or replace every day.
2. After 7-10 days of culture in vascular differentiation medium, purify CD31⁺ or CD34⁺ vascular cells from the adherent monolayer with a magnetic bead cell sorting method.
   ​NOTE: Each individual clone may display distinct kinetics of differentiation. Determine peak timepoint for CD31/CD34 expression by FACS analysis (Figure 4A). Pre-, and post- magnetic bead enrichment enrichment FACS analysis is required. Hematopoietic progenitors (HP) may be recovered at this stage from the supernatant and enriched using CD34 microbeads. CD34⁺ XF-hiPSC-derived HP may be further validated using methylcellulose assay (Figure 4B-C)¹⁶.
   1. In a biosafety cabinet, aspirate culture medium and wash cells in PBS (2 mL per well). Aspirate PBS and dissociate hiPSC colonies into single cells by adding enzymatic dissociation reagent (e.g., Accutase, 1 mL per well). Incubate the cells for 5 min at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere).
   2. In a biosafety cabinet, collect cells by adding an equal volume of culture medium. Centrifuge cells at 200 x g for 5 min.
   3. Resuspend cell pellet in vascular differentiation medium (Table 2). Count the number of cells using a hemocytometer or an automatic cell counter. Set aside about 50,000-100,000 cells for pre-magnetic bead enrichment FACS assay using directly conjugated anti-CD31 or anti-CD34 antibody.
   4. Centrifuge the remaining cells at 300 x g for 5 min. Resuspend cell pellet in magnetic bead enrichment buffer (PBS pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA) in a biosafety cabinet.
   5. Pass the cells through a cell strainer (40-70 µm filter). Centrifuge at 300 x g for 5 min. Resuspend in 60 µL magnetic bead enrichment buffer.
   6. Add 20 µL FcR Blocking Reagent, then add 20 µL CD31 or CD34 microbeads and incubate for 15-20 min at 4 °C.
   7. Wash cells with 2 mL magnetic bead enrichment buffer in a biosafety cabinet and centrifuge (300 x g, 5 min).
   8. Resuspend cell pellet in 1 mL magnetic bead enrichment buffer in a biosafety cabinet.
   9. Place an LS Column in the magnetic field of a magnetic cell separator within the biosafety cabinet. Wash column with 3 mL magnetic bead enrichment buffer.
   10. Apply cells to column.  Collect unlabeled cells into a 15 mL conical tube. Wash the column 3 times with 3 mL magnetic bead enrichment buffer.
   11. Remove the column from the separator and place it onto a new 15 mL conical tube.
   12. Flush out the magnetically labeled cells by adding 3-5 mL endothelial growth medium. Centrifuge at 200 x g for 5 min.
   13. Resuspend in 1 mL endothelial growth medium in a biosafety cabinet. Count the number of cells using a hemocytometer or an automatic cell counter. Set aside about 50,000 cells for post-magnetic bead enrichment sort purity by FACS analysis using directly conjugated anti-CD31 or anti-CD34 antibody.
   14. Isolated VP can be further expanded on fibronectin-coated (10 µg/mL) tissue culture-treated plates of flasks in endothelial growth medium.

6. Experimental design guidelines for functional validation of XF-hiPSC

1. Establish stocks of validated XF-hiPSC at the earliest passages using an established non-integrating method, such as episomal^7,17, Sendai virus¹⁸ or mRNA¹⁹ reprogramming.
   NOTE: A comparison of the efficiency and reliability of non-reprogramming methods is provided elsewhere⁶. Transgenic material clearance in episomal or Sendai virus reprogramming methods can be verified during initial passages using PCR methods^7,20. Transgene retention is not compatible with TIRN-SC generation.
2. Validate normal karyotype in XF-hiPSC clones by G-banding or whole-genome array-based coverage assays⁹.
3. Validate gene expression and epigenomic integrity of XF-hiPSC lines (i.e., parental imprints, X activation) using DNA methylation and gene expression arrays or sequencing methods^5,11,14.
4. Validate basic functional pluripotency using teratoma differentiation protocols (Figure 2E), quantitative embryoid body (EB) differentiation assays using RT-PCR arrays¹⁴, or directed differentiation assays toward the 3 germ layers⁹.
   NOTE: Quantitative methods for multilineage differentiation capacity and intra-lineage proliferative index of teratoma assays using histological characterization by microscopy are provided elsewhere⁴. Protocols for directed differentiation toward mesodermal lineages and hemato-vascular specification (Figure 4A-C) were previously detailed^4,5,9, as well as directed differentiation assay toward definitive endoderm (Figure 4D)⁵. Examples of directed differentiation assay toward ectoderm are provided elsewhere^4,5.

7. Chemical TIRN adaptation of XF-hiPSC

1. Purchase or prepare in-house low-passage MEF feeders from CF1 or CF1 x DR4 hybrid E13.5 mouse embryos following published protocols^9,21. Plate 200,000 MEF feeders per well in a gelatinized (0.1% gelatin in water, 1 mL per well) tissue culture-treated 6-well plate at least one day before TIRN adaptation step (detailed TIRN protocols are provided elsewhere^4,5,9).
2. Maintain and expand transgene-free XF-hiPSC cultures with validated normal karyotypes in a feeder-free culture system (e.g., E8 medium, see Table 1 for in-house formulation).
3. Replace E8 medium with LIF-5i medium (2 mL per well; Table 1) after XF-hiPSC cultures have reached ~50% confluency (i.e., 3-5 days after initial plating) in a biosafety cabinet.
4. The next day, aspirate culture medium and wash LIF-5i-adapted XF-hiPSC once with PBS (2 mL per well) in a biosafety cabinet. Aspirate supernatant and add 1 mL of enzymatic cell dissociation reagent to each well. Incubate for 5 min at 37 °C in a CO₂ incubator.
5. Gently triturate with a pipette into a single cell suspension in a biosafety cabinet. Neutralize with LIF-5i medium (1mL per well). Collect the cell suspension into a sterile 15 mL conical tube.  Centrifuge at 200 x g for 5 min.
6. Aspirate the supernatant in a biosafety cabinet. Resuspend the cell pellet in 2 mL of LIF-5i medium.
   NOTE: If possible, take an aliquot for cell counting. It is possible to neutralize enzymatic activity with LIF-5i/LIF-3i medium without supplementation with inhibitors or growth factors.
7. Wash the MEF plate twice with PBS (2 mL per well in 6-well plates) and add cells (1 well-equivalent of LIF-5i adapted cells onto 1 well of MEF feeders in 2 mL LIF-5i medium).
   NOTE: Optimization of initial plating densities for each individual XF-hiPSC line is optional. If cells were counted in the previous step, plate a minimum of 1x10⁶ cells per well onto feeders.  Transfer the plate in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere).
8. Culture and maintain XF-hiPSC for 3-5 days in LIF-5i on MEF in a CO₂ incubator (37 °C, 5% CO₂, humid atmosphere). Change LIF-5i medium daily.
9. When dome-shaped TIRN-SC colonies are formed (after 3-5 days of culture on feeders), aspirate culture medium in a biosafety cabinet. Wash each well  with 2 mL of PBS.
10. Aspirate PBS. Add 1 mL of cell detachment solution (e.g., Accutase) to each well. Incubate for 5 min at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere).
11. Add 1 mL of LIF-3i medium (Table 1) to each well in a biosafety cabinet to neutralize the dissociation reagent. Gently triturate and collect cells into a 15 mL conical tube. Centrifuge at 200 x g for 5 min.
12. Aspirate the supernatant in a biosafety cabinet. Re-suspend in LIF-3i medium (1 mL per well) for cell counting using a hemocytometer or an automatic cell counter. Plate ~5 x 10⁵ cells in 2 mL LIF-3i medium per well onto irradiated MEF in gelatinized 6-well plates in a biosafety cabinet.
    NOTE: repeat steps 7.12 5o 7.16 for all subsequent passages.  Following the initial LIF-5i-adapted culture, plate LIF-3i cultures at a lower density (2x10⁵ cells/well) and passage every 3-4 days.
13. Passage TIRN-reverted XF-hiPSC for at least 6-7 continuous passages in LIF-3i medium prior to use in functional studies or cryopreservation. Record the number of passages in either xenofree primed culture medium (e.g., E8) or LIF-5i/LIF-3i TIRN media.
    NOTE: TIRN reversion of high-passage (e.g., >p40) XF-hiPSC lines is inefficient and not recommended. High-passage lines often harbor chromosomal or sub-chromosomal defects, which are not compatible with efficient TIRN reversion, and will result in poor reversion efficiencies and limited culture stability. Also, the use of TIRN cultures that have undergone greater than 15 passages in LIF-3i medium is not recommended for functional studies. The functional impact of TIRN reversion of XF-hiPSC is most prominent between 7 and 15 passages in LIF-3i medium.

8. Experimental design guidelines for directed differentiation of XF/FF hiPSC-derived TIRN-SC.

1. Directly utilize XF/FF hiPSC-derived TIRN-SC into established directed differentiation protocols without any "re-priming" step (i.e., no requirement for switching back TIRN stem cells to conventional “primed” conditions prior to their use in directed differentiation assays).
2. For functional evaluation of TIRN impact onto XF-hiPSC, set up sibling isogenic cultures, at equivalent passage number in parallel. Maintain primed/TIRN sibling isogenic XF-hiPSC cultures in parallel in their respective media (e.g., E8 on vitronectin vs. LIF-3i on MEF), and simultaneously perform teratoma assay (Figure 5), or differentiate using identical embryoid body or directed differentiation protocols and materials (Figure 6).
3. Perform functional evaluation of terminal differentiated lineages, when possible. For instance, vascular progenitors can be evaluated by protein expression, DiI-conjugated acetylated low-density lipoprotein (LDL) uptake assay and Matrigel tube assay (Figure 7).
   NOTE: Additional functional assays for CB-hiPSC- and TIRN-SC-derived VP (i.e., DiI-Ac-LDL uptake/senescence/proliferation/migration assays, in vitro Matrigel tube assays, animal Matrigel plugassay, xenotransplantation assays) are detailed elsewhere^3,4.
4. Adjust and optimize initial plating densities and kinetics (Figure 6) for each individual XF-hiPSC clone in each differentiation assay. 
   NOTE: TIRN-reverted XF-hiPSC have a more robust proliferative and differentiation capacity in directed differentiation assays than primed XF-hiPSC.TIRN cultures typically require a lower initial plating density than E8 cultures, and do not require the use of anti-apoptotic supplements to enhance clonal survival.

Results

This protocol optimizes published methods for efficient myeloid progenitor reprogramming to a high-fidelity primed pluripotent state using episomal vectors and stromal signals⁷. The protocol (Figure 1) outlines defined methods for XF/FF stromal-primed episomal reprogramming of human CB CD34⁺ progenitors into XF-hiPSC, and instructs how to validate their functionality via directed hemato-vascular specification, followed by TIRN reversion to blastome...

Discussion

The Zambidis group originally reported a highly optimized four-factor episomal method for reprogramming lineage-committed CB myeloid progenitors into CB-hiPSC with bulk efficiencies reaching up to 50% in purified episome-expressing cells⁷. Herein, we presented a modified version of the original human CB progenitor episomal reprogramming protocol in XF/FF stroma-primed conditions. This method employed the augmented reprogramming capacity of lineage-committed CD33⁺CD45⁺CD34

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amaxa Biosystems Nucleofector II	Lonza	AAB-1001
anti-CD143 (BB9) antibody, APC conjugated	BD Biosciences	557929	use 10-20 µL per assay (FACS)
anti-CD144 antibody, PE conjugated	BD Biosciences	560410	use 10-20 µL per assay (FACS)
anti-CD146 antibody, PE conjugated	BD Biosciences	550315	use 10-20 µL per assay (FACS)
anti-CD184/CXCR4 antibody, APC conjugated	BD Biosciences	555976	use 10-20 µL per assay (FACS)
anti-CD31 antibody	BD Biosciences	550389	dilute 1:100 in blocking solution for immunofluorescent assay
anti-CD31 antibody, APC conjugated	eBioscience	17-0319-42	use 2 µL per assay (FACS)
anti-CD34 antibody, APC conjugated	BD Biosciences	555824	use 5 µL per assay (FACS)
anti-CD45 antibody, PE conjugated	BD Biosciences	555483	use 10-20 µL per assay (FACS)
anti-NANOG antibody, PE conjugated	BD Biosciences	560483	use 10-20 µL per assay (FACS)
anti-OCT4 antibody, PE conjugated	R&D Systems	IC1759P	use 10 µL per assay (FACS)
anti-SOX17 antibody, Alexa488 conjugated	BD Biosciences	562205	use 5 µL per assay (FACS)
anti-SOX2 antibody, PE conjugated	R&D Systems	IC2018P	use 10 µL per assay (FACS)
anti-SSEA-4 antibody, APC conjugated	R&D System	FAB1435A	use 5 µL per assay (FACS)
anti-SSEA-4 GloLIVE antibody, NL493 conjugated	R&D System	NLLC1435G	use at 1:50 dilution (live and fixed immunostainings)
anti-TRA-1-60 A488 conjugate kit	ThermoFisher Scientific	A25618
anti-TRA1-60 antibody, PE conjugated	BD Biosciences	560193	use 10 µL per assay (FACS)
anti-TRA-1-60 GloLIVE antibody, NL557 conjugated	R&D Systems	NLLC4770R	use at  a 1:50 dilution (live and fixed immunostainings)
anti-TRA-1-60 StainAlive Antibody, DyLight 488 conjugated	Stemgent	09-0068	use at a 1:100 dilution (live and fixed immunostainings)
anti-TRA1-81 antibody, PE conjugated	BD Biosciences	560161	use 10 µL per assay (FACS)
anti-TRA-1-81 StainAlive Antibody, DyLight 488 conjugated	Stemgent	09-0069	use at a 1:100 dilution (live and fixed immunostainings)
CD31 MicroBead Kit, human	Miltenyi Biotec	130-091-935
CD34 MicroBead Kit, human	Miltenyi Biotec	130-046-702
CD34+ Mixed Donors,  Cord blood, 1 million cells	AllCells	CB, CR, CD34+, Mixed, PS, CS10, 1M	alternative sources of cells include fetal liver, adult bone marrowand mobilized peripheral blood
CF1 mouse	Charles river	023
CHIR99021	R&D System	L5283	reconstitute at 100 mM in DMSO
Corning Costar tissue culture-treated 6-well plates	Corning	3506
Corning Synthemax II-SC Substrate, 10 mg Vial	Corning	3535	use at 5 μg/cm2 as a replacement for Corning 3978
Corning Synthemax-R 6-well plate	Corning	3978	discontinued and replaced to 3535
Costar 6-well Clear Flat Bottom Ultra-Low Attachment 6-well plate	Corning	3471
Countess  cell counting chamber slide	Thermo Fisher Scientific	C10228
Countess automated cell counter	Thermo Fisher Scientific	AMQAX1000
CryoStore CS10	StemCell Technologies	100-1061
DII-Ac-LDL	Thermo Fisher Scientific	L3484	use at 5-15 µg/mL
DMEM (Dulbecco's Modified Eagle Medium)	Thermo Fisher Scientific	11995065
DMEM/F-12 , GlutaMAX supplement	Thermo Fisher Scientific	10565018
DMEM/F-12, HEPES	Thermo Fisher Scientific	11330032
DMSO (dimethyl sulfoxide)	Sigma Aldrich	D2650
DR4 mouse	The Jackson Laboratory	3208
Essential 8 (E8) medium	StemCell Technologies	5940
Fetal bovin serum (FBS)	Thermo Fisher Scientific	SH30071.03
FIX & PERM Cell Permeabilization Kit	Thermo Fisher Scientific	GAS004
Forskolin	Stemgent	04-0025	reconstitute at 100 mM in DMSO
Gelatin (porcine)	Sigma Aldrich	G1890-100G	resuspend in water and sterilize with an autoclave
human CD34+ cell nucleofector kit	Lonza	VPA-1003
Isotype mouse IgG1 PE conjugated	BD Biosciences	554680
Isotype mouse IgG2a PE conjugated	BD Biosciences	558595
Isotype rat IgG2b PE conjugated	R&D Systems	IC013P
KnockOut Serum Replacement	Thermo Fisher Scientific	10828-028
L-Ascorbic acid	Millipore Sigma	A8960
L-Glutamine (100X)	Thermo Fisher Scientific	25030-081
LS columns	Miltenyi Biotec	130-042-401
Matrigel	Corning	356237	use 200 µL/well in 48-well plates
MEM Non-essential amino acid (MEM NEAA) (100X)	Thermo Fisher Scientific	11140-050
MethoCult SF H4436	StemCell Technologies	04436
MidiMACS Separator	Miltenyi Biotec	130-042-302
mTeSR1 medium	StemCell Technologies	85850
Nalgene cryogenic vials	Thermo Fisher Scientific	5000-0020
Paraformaldehyde Solution, 4% in PBS	Thermo Fisher Scientific	J19943.K2	dilute to the required concentration using PBS
PD0325901	Sigma Aldrich	PZ0162	reconstitute at 100 mM in DMSO
Penicillin/streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140-122
pEP4 E02S EM2K episome	Addgene	20923	Plasmids were propagated in TOP10 E. coli (Invitrogen) and purified with QIAGEN plasmid Maxi kits.
Phosphate buffered saline (PBS)	Biological Industries	02-023-1A
Poietics human mesenchymal stem cells	Lonza	PT-2501	750,000 cells at passage 2 can be thawed and expanded according to manufacturer's instructions for 1-2 passages before use or secondary banking
Purmorphamine	Stemgent	04-0009	reconstitute at 10 mM in DMSO
recombinant Activin A	Peprotech	120-14E	resuspend at  (100 µg/mL) in sterile PBS, 0.1% human or bovine serum albumin
recombinant human FGF2	Peprotech	100-18B	resuspend at  (100 µg/mL) in sterile PBS, 0.1% human or bovine serum albumin
recombinant human FGF-basic (bFGF)	Peprotech	100-18B	resupend at 100 µg/mL in 0.1% bovine serum/human albumin in PBS
recombinant human FLT3 ligand	R&D Systems	308-FKHB or  308E-GMP	resuspend at  (100 µg/mL) in sterile PBS, 0.1% human or bovine serum albumin
recombinant human Insulin	Biogems	10-365
recombinant human Kit-ligand	R&D Systems	BT-SCF or BT-SCF-GMP	resuspend at  (100 µg/mL) in sterile PBS, 0.1% human or bovine serum albumin
recombinant human LIF	Peprotech	300-05	resupend at 100 µg/mL in 0.1% bovine/human serum albumin in PBS
recombinant human TGFβ1	Peprotech	100-21	resupend at 100 µg/mL in 0.1% bovine/human serum albumin in PBS
recombinant human thrombopoietin	R&D Systems	288-TPN or 288E-GMP	resuspend at  (100 µg/mL) in sterile PBS, 0.1% human or bovine serum albumin
recombinant human transferrin	Millipore Sigma	T3705
recominant human BMP4	Peprotech	120-05	resuspend at  (100 µg/mL) in sterile PBS, 0.1% human or bovine serum albumin
SB431543	Selleckchem	S1067	reconstitute at 10 mM in DMSO
SIGMAFAST, alkaline phosphatase substrate, chromogenic, tablet	Millipore Sigma	B5655	dissolve 1 tablet in 10 mL of purified water
Sodium selenite	Millipore Sigma	S5261
Stemolecule Y27632 in Solution	Stemgent	04-0012-02	ROCK inhibitor in solution (10 mM)
StemPro Accutase Cell Dissociation Reagent	Thermo Fisher Scientific	A11105-01
StemPro MSC SFM XenoFree (XF-MSC)	Thermo Fisher Scientific	A1067501	Prepare XF-MSC medium by mixing 5mL StemPro MSC SFM XenoFree Supplement with 500 mL StemPro MSC SFM Basal Medium. Aliquoted medium can be cryopreserved at -20°C.
StemSpan SFEM	StemCell Technologies	9600
Thermo Scientific Mr. Frosty Freezing Container	Thermo Fisher Scientific	5100-0001
Ulex Europaeus Agglutin I (UEA-1), biotynilated	Vector Laboratories	B-1065-2	dilute 1:200 in blocking solution for fluorescent assay
VEGF	Peprotech	100-20	resuspend at  (100 µg/mL) in sterile PBS, 0.1% human or bovine serum albumin
Versene solution	Thermo Fisher Scientific	15040066
Vitronectin XF matrix	StemCell Technologies	7180	dilute at 40 µL/mL in CellAdhere dilution buffer
XAV939	Sigma Aldrich	X3004	reconstitute at 100 mM in DMSO
β-mercaptoethanol	Thermo Fisher Scientific	21985-023	light sensitive