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 reprogramm....

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 Ani....

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