HLA-homozygous hiPSC haplobank for the Spanish population: generation, GMP expansion and population coverage (Kuebler et al., 2023)

Background

  • Stem cell therapies using induced pluripotent stem cells (iPSCs) offer a route to regenerative medicine and have potential for broad application. A key cost driver is generating patient-specific iPSCs; an alternative is allo­genic, homozygous HLA iPSC lines to create a clinical-grade haplobank that covers a large fraction of the population.

  • The study reports the generation of the first clinical-grade, HLA-homozygous iPSC bank (haplobank) in Spain using cord blood units (CBUs) that are homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes.

  • Source material: CD34+ hematopoietic progenitors isolated from frozen CBUs; these CD34+ cells were reprogrammed using Sendai virus vectors to iPSCs under good manufacturing practice (GMP) conditions.

  • Aim: to create master cell banks (MCBs) and working cell banks (WCBs) of seven haplotyped iPSC lines, expanded and banked under GMP, to support advanced therapy medicinal product (ATMP) development and off-the-shelf cell therapies.

  • This haplobank is intended as starting material for ATMP development and as a foundation for European/international haplobanks through shared haplotype resources.

  • Licensing and ethics: All donor material was used under project-specific informed consent, with ethics approval and adherence to regulatory guidelines.

Key concepts and definitions

  • iPSC: human-induced pluripotent stem cells; somatic cells reprogrammed to a pluripotent state.

  • Haplobank: a bank of iPSC lines that are homozygous for common HLA haplotypes to maximize population coverage and minimize immune rejection in allogeneic therapies.

  • HLA haplotypes: combinations of alleles at HLA-A, HLA-B, and HLA-DRB1 (and related loci) that determine immune compatibility.

  • CD34+ cells: hematopoietic progenitors used as starting material for reprogramming to hiPSCs.

  • GMP (Good Manufacturing Practice): regulatory framework ensuring quality and safety in manufacturing biologics and cell therapies.

  • ATMP (Advanced Therapy Medicinal Product): regulatory category for cell- and gene-based therapies.

  • Sendai virus (SeV) reprogramming: non-integrating viral method used to reprogram somatic cells to iPSCs; transient and eventually cleared from cells.

  • MCB/WCB: Master Cell Bank and Working Cell Bank; layers of cell banking used for consistent manufacturing.

  • QC (Quality Control): a set of assays to ensure sterility, purity, identity, genetic stability, and absence of contaminants.

  • STR (Short Tandem Repeat) profiling: genetic fingerprinting to confirm cell line identity.

  • EB (Embryoid Body) formation: in vitro assay to test differentiation potential into three germ layers.

  • 0MM, 1MM, 2MM: zero, one, or two mismatches tolerated in matching at the HLA loci during coverage calculation.

Haplobank design and population coverage

  • Donor source and haplotype selection:

    • Cord blood units were screened from the Spanish Stem Cell Transplant Registry for homozygosity at the most common HLA haplotypes (HLA-A, HLA-B, HLA-DRB1).

    • Seven donors were selected to maximize population coverage of Spain, achieving 21.37% coverage under zero-mismatch (0MM) criteria.

    • The haplotypes provide the widest possible coverage of the Spanish population based on the dataset used.

    • Donors’ cord blood units were quality verified and donors provided informed consent for the use of CB units to generate hiPSC haplolines.

  • Population coverage calculation:

    • Population cohort: combined data from adult bone marrow donors and cord blood donors in REDMO, at four-digit resolution for 56{,}798 individuals.

    • Method: iterative, in-house algorithm in Microsoft Access. Brief steps:

    • Step 1: Check the first haplotype against the whole cohort and extract all matched individuals.

    • Step 2: Recompute coverage for the next haplotype using the remaining data.

    • Step 3: Repeat across haplotypes, accumulating matches.

    • Step 4: To compute “beneficial match” (allowing 1 or 2 mismatches in any locus), repeat the same strategy but tolerate one or two mismatches.

    • Coverage metric:

    • For any given level of mismatch allowance, cumulative coverage = (matched individuals in all iterations) / N × 100, where N = 56{,}798.

    • Reported coverage for the seven haplotypes (Spain):

    • 0MM (no mismatches): 21.37 ext{%}

    • 1MM (allowing one mismatch): 50.83 ext{%}

    • 2MM (allowing two mismatches): 92.46 ext{%}

  • Haplotypes and donor details (Table 1 overview):

    • Seven haplo-homozygous haplotype lines are designated as CBiPS1–CBiPS8, each associated with a donor and specific HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DPB1 alleles; ABO blood group and donor sex are recorded.

    • Examples of haplotype lines and their donor metadata were presented (precise allele values per line are provided in the study’s Table 1).

  • Rationale and context:

    • Cord blood was chosen because it is routinely collected, HLA-typed, has low risk to mother/child at collection, and harbors hematopoietic progenitors suitable for iPSC generation.

    • CB banks often retain surplus units suitable for iPSC derivation; cost and time reductions are expected with haplobanks.

  • Ethical/regulatory context:

    • CBUs were provided from the clinical inventory with reconsenting per IPS-PANIA project; ethics committee approval (ID ethics committee: PR(AG)428/2018).

    • The haplobank is intended to serve as starting materials for ATMP development under GMP, ensuring traceability and regulatory compliance.

Donor selection and cord blood processing

  • Donor and sample selection:

    • CB donors homozygous for the most frequent HLA-A, HLA-B, and HLA-DRB1 haplotypes were chosen to maximize coverage of the Spanish population.

    • Donors signed consent for use of donated CB for hiPSC haplotype derivation.

  • Cord blood processing:

    • CB units thawed, osmotically equilibrated, and mononuclear fraction isolated by Ficoll density gradient in closed Sepax2 system.

    • CD34+ cells isolated using clinical-grade anti-CD34 magnetic beads and magnetic columns (Miltenyi Biotec).

Reprogramming and hiPSC generation

  • CD34+ cell expansion:

    • Expanded in StemPro 34 Serum Free Medium with cytokines for 4 days: 50 ng/mL SCF, 50 ng/mL FLT3L, 10 ng/mL TPO, 10 ng/mL IL-6; 37°C, 5% CO2; medium changed every other day.

  • Reprogramming with Sendai virus kits:

    • Transduction with CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit at 1×10^4 cells per transduction in SP34 SFM with cytokines and polybrene.

    • Residual CD34+ cells cryopreserved; next day, infected cells seeded on Biolaminin-521 CTG-coated dishes in SP34 SFM with cytokines.

    • Day 6: switch to Essential 8 Flex Medium; monitor for colonies; emergence of reprogrammed colonies typically around 16–18 days after infection.

    • Colonies picked and seeded on CTG laminin-coated dishes in Essential 8 Flex (+ RevitaCell) for expansion.

    • Upon colony rise and single-clone picking, Sendai virus-infected cells were frozen for later testing.

    • Absence of Sendai virus confirmed by RT-PCR: RNA extracted, cDNA synthesized, PCR performed to detect Sendai genome; positive control included.

    • Genomic integrity check by G-banding karyotype performed on two clones; one virus-free clone with normal karyotype selected per line for expansion.

    • Objective: produce clones free of Sendai virus and transgenes with euploid karyotype.

Culture, cryopreservation, and clone selection

  • hiPSC culture and cryopreservation:

    • Clones passaged with CTS DPBS (no Ca/Mg) and CTS Versene; seeded on CTG LN-521-coated plates in Essential E8 Flex.

    • Cryopreservation: cells pelleted, resuspended in CTS PSC Cryo-medium with RevitaCell, frozen at −80°C, then transferred to liquid nitrogen.

  • Clone selection criteria:

    • Absence of Sendai virus genome and transgenes confirmed by RT-PCR around passages 7–8 with 15 days at 39°C.

    • Normal karyotype confirmed by G-banding.

    • One clone per line selected for full characterization and banking after screening 2 virus-free clones.

GMP generation of MCBs and WCBs; downstream QC

  • GMP expansion to create MCBs and WCBs:

    • MCBs produced under GMP in classified facilities in a clean room with GMP-grade reagents.

    • Previous steps were assessed for sterility (BacTAlert), mycoplasma (qPCR), and adventitious viruses.

    • For banking: two vials per line, thawed and expanded to 70–80% confluence, then split into 6–8 plates per line; after expansion, cells cryopreserved in 40–50 vials with ≥1×10^6 viable cells per vial.

    • After 24 h at −80°C, cryovials moved to LN2 storage.

  • WCB production:

    • Two MCB vials thawed, expanded, and frozen following the same procedure as MCB.

  • Characterization workflow (applied to MCB and WCB):

    • Immunocytochemistry (ICC) for pluripotency markers (Nanog, OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4).

    • Alkaline phosphatase (AP) staining.

    • In vitro differentiation via embryoid body (EB) formation and ICC for endoderm, ectoderm, and mesoderm markers.

    • Short tandem repeats (STR) analysis for identity.

    • Mycoplasma testing at early passages and before banking; karyotyping repeated after banking.

  • MCB/WCB quality controls:

    • Sterility (Ph. Eur. 2.6.27), mycoplasma (2.6.7), endotoxin (2.6.14), adventitious virus testing.

    • Viability before freezing and recovery after thawing (7AAD viability assay and colony-forming ability after thaw).

    • Acceptance criteria included viable cell numbers, recovery, and confluence metrics as per Table 3 (details below).

Characterization and identity testing

  • Immunocytochemistry for pluripotency:

    • Markers tested: Nanog, OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4.

  • Differentiation capacity:

    • EB differentiation to three germ layer markers: endoderm (AFP, FOXA2), ectoderm (TUJ1, GFAP), mesoderm (ASMA, GATA4).

  • Identity and genetic stability:

    • STR analysis showed iPSC line STRs identical to the parental CB CD34+ cells.

    • Karyotyping confirmed euploid status; lines 3×46,XX and 4×46,XY across lines.

  • Registration and traceability:

    • All seven iPSC lines registered with the Spanish National Stem Cell Bank and the European human pluripotent stem cell registry (hPSCReg).

  • Supplementary data:

    • Supplementary information contains additional figures validating Sendai virus absence (Fig. S1) and other QC data (Figs. S2–S3, Tables S2–S3).

Detailed results for the seven haplotype lines

  • Recurrent data points per line (Table 2 overview):

    • For each donor-derived hiPSC line, expansion of CD34+ cells from the CB unit was quantified at day 1 and day 4, showing expansion in the range from ~0.23×10^6 to ~3.46×10^6 cells and day-4 expansions up to ~3.46×10^6 cells.

    • Expansion factor (day4/day1) ranged from ~1.45 to ~3.87 depending on line.

    • Reprogramming efficiency per line ranged roughly from ~0.45% to ~1.8% (values shown in Table 2).

  • From each line:

    • Between 10 and 14 clones were initially picked and expanded; five clones per line were selected for further analysis.

    • From these five clones, RNA was extracted and tested to confirm absence of Sendai virus signals; two virus-free clones per line were subjected to karyotype analysis.

    • Finally, one virus-free clone per line with normal karyotype was chosen for full characterization and banking.

  • Table 1 (haplotype details): seven haplotypes (CBiPS1–CBiPS8) each mapped to specific HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DPB1 alleles; ABO type and sex recorded.

    • Example haplotype entries include (illustrative formatting):

    • CBiPS1-Sv4F-B8: HLA-A 29:02, HLA-B 44:03, HLA-C 16:01, HLA-DRB1 07:01, HLA-DQB1 02:02, HLA-DPB1 11:01; ABO O+, Sex XY.

    • CBiPS2-Sv4F-D10: HLA-A 30:02, HLA-B 18:01, HLA-C 05:01, HLA-DRB1 03:01, HLA-DQB1 02:01, HLA-DPB1 02:02/04:01; ABO O+, Sex XY.

    • CBiPS3-Sv4F-E9: HLA-A 03:01, HLA-B 07:02, HLA-C 07:02, HLA-DRB1 15:01, HLA-DQB1 06:02, HLA-DPB1 02:01/04:01; ABO O+, Sex XY.

    • CBiPS4-Sv4F-F6: HLA-A 01:01, HLA-B 08:01, HLA-C 07:01, HLA-DRB1 03:01, HLA-DQB1 02:01, HLA-DPB1 01:01/03:01; ABO O-, Sex XX.

    • CBiPS6-Sv4F-H6: HLA-A 33:01, HLA-B 14:02, HLA-C 08:02, HLA-DRB1 01:02, HLA-DQB1 06:02, HLA-DPB1 02:01P; ABO O-, Sex XX.

    • CBiPS7-Sv4F-I12: HLA-A 24:02, HLA-B 07:02, HLA-C 07:02, HLA-DRB1 15:01, HLA-DQB1 06:02, HLA-DPB1 04:01P; ABO O+, Sex XY.

    • CBiPS8-Sv4F-J1: HLA-A 11:01, HLA-B 27:05, HLA-C 01:02, HLA-DRB1 01:01, HLA-DQB1 05:01, HLA-DPB1 04:01/11:01; ABO O+, Sex XX.

  • Karyotype and pluripotency:

    • All seven haplolines were euploid (3 lines 46,XX; 4 lines 46,XY) at clone selection (passages 9–16) and banking (passages 13–17).

    • All seven lines stained positive for AP and expressed pluripotency markers (Nanog, OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4).

    • In vitro differentiation demonstrated capacity for endoderm (AFP, FOXA2), ectoderm (TUJ1, GFAP), and mesoderm (ASMA, GATA4).

    • STR analysis confirmed identity with the corresponding CB CD34+ source.

  • Banking status:

    • All seven haplolines were registered with the Spanish National Stem Cell Bank and with the European registry (hPSCReg).

  • Figure and table references (in the study):

    • Fig. 1 shows cumulative haplotype coverage of the Spanish population with 0MM, 1MM, and 2MM scenarios.

    • Fig. 2 and Fig. 3 depict morphology and immuno-detection of pluripotency and differentiation markers.

    • Table 3 presents the quality control assays and acceptance criteria for GMP-MCB and GMP-WCB.

    • Supplementary figures (S1–S3) provide additional QC data, including proof of absence of Sendai virus.

Comparative context and discussion

  • Context of haplobanking in other populations:

    • Prior work in Japanese, Korean and European populations shows that relatively small numbers of haplotypes can cover substantial portions of populations, with larger haplotypes banks needed for higher coverage.

    • Nakatsuji et al. and Okita et al. demonstrated that a targeted set of homozygous haplotypes can cover large fractions of the population, but scale and donor availability vary by population.

    • CiRA (Japan) and Korea’s KHIB have produced substantial haplobanks, with several dozen lines covering a portion of their populations.

  • Population coverage implications:

    • For Spain, the seven haplo-lines achieve 21.37% coverage with 0MM, 50.83% with 1MM, and 92.46% with 2MM, highlighting that increasing the mismatch tolerance dramatically increases population coverage.

    • In some clinical scenarios, less stringent HLA matching may be acceptable with immunosuppression, enabling broader coverage.

  • Global/regulatory perspective:

    • Haplobanks aim to reduce manufacturing costs and immune rejection risk by providing off-the-shelf iPSC-derived products for multiple indications.

    • The regulatory pathway classifies iPSC derivatives as ATMPs; GMP-compliant production and traceability are essential.

    • GMP adaptation: some haplotype iPSC lines are generated under GMP or are adapted to GMP after initial generation; many current clinical trials use non-GMP lines subsequently qualified for GMP.

  • Ethical and governance considerations:

    • Donor consent, re-consenting for research use, and ethics approvals are essential; ongoing donor engagement may be required.

    • International coordination (e.g., COST Action HAPLO-iPS) aims to standardize methodologies, data sharing, and training, and to integrate haplobanks into registries such as hPSCReg.

  • Practical implications for therapy development:

    • Haplobanks provide a scalable, cost-reducing platform for generating derivatives of a wide array of tissues (retina, heart, neural tissue, blood cells, etc.).

    • The study emphasizes that iPSCs should be used as an intermediate production step under strict QC to ensure comparability across lines and facilities.

  • Limitations and future directions:

    • The approach relies on donor availability and continued validation of genetic stability across passaging and banking.

    • Further standardization of QC assays and regulatory guidance is necessary to harmonize cross-border use of haplobanks.

  • Regulatory and scientific takeaway:

    • The haplobank represents a strategic approach to off-the-shelf cell therapies, balancing immunogenetics, manufacturing feasibility, and clinical utility within a European context.

Conclusions and implications for ATMP development

  • The haplobank described here constitutes a valuable intermediate cell bank capable of covering >20% of the Spanish population for 0MM HLA matching, with substantial additional coverage when mismatches are permitted (up to ~92% for 2MM).

  • These haplolines, generated from seven CBUs homozygous for common HLA haplotypes, provide a scalable starting material for ATMP development and may be shared across Europe and beyond due to haplotype sharing.

  • The work highlights the feasibility of producing GMP-compliant MCBs/WCBs from CB-derived hiPSCs, with rigorous QC, donor consent, and traceability to support clinical applications.

  • The initiative supports a coordinated European framework (e.g., COST action, hPSCReg) and aligns with global efforts to establish national haplobanks for safer, faster, and more cost-effective cell therapies.

Practical details and takeaways for exam preparation

  • Key workflow sequence:

    • Identify homozygous CBUs with common Spanish haplotypes → isolate CD34+ progenitors → expand in defined cytokine-supplemented medium → reprogram with Sendai CytoTune kit → select virus-free iPSC clones with normal karyotype → expand under GMP → bank MCBs and WCBs → perform QC and characterize lines → register lines.

  • Major outcomes to remember:

    • 7 haplo-homozygous CBUs yielded 7 hiPSC lines (CBiPS1–CBiPS8) with euploid karyotypes and pluripotency markers.

    • Population coverage of Spain: 0MM = 21.37%, 1MM = 50.83%, 2MM = 92.46%.

    • All lines were banked under GMP with MCBs and WCBs tested for sterility, mycoplasma, endotoxin, and adventitious viruses; identity confirmed by STR; regulatory registration in national and European registries.

  • Important regulatory concepts:

    • iPSC derivatives used for cell therapy are considered ATMPs and require GMP manufacturing, traceability, and robust QC.

    • The study shows how to balance donor selection, haplotype coverage, and GMP requirements to build a clinically useful haplobank.

Abbreviations

  • ATMP: Advanced Therapy Medicinal Product

  • BMI: (not used here; see context) – ignore if not relevant

  • BAC: (not used here; see context) – ignore if not relevant

  • A list of abbreviations used in the paper includes:

    • ATMP, BM, CB, CBU, CTS, CTG, EMA, EB, FDA, GMP, hiPSC, HLA, ICC, iPSC, MCB, QC, REDMO, STR, WCB, WMDA, etc.

Supplementary information and references

  • The article provides extensive supplementary data including details on: absence of Sendai virus in clones (Additional Fig. S1), additional QC data (Figs. S2–S3, Table S2–S3).

  • Funding and ethics: Project IPS-PANIA funded by MCIN; ethics committee approval; donor informed consent. The COST Action and hPSCReg integration are highlighted for future European-scale haplobank collaboration.

Summary statement

  • This study documents the generation of the first clinical-grade, HLA-homozygous iPSC haplobank in Spain, derived from seven cord blood units homozygous for common haplotypes, with GMP-compliant expansion and banking, comprehensive characterization, and robust QC. The resulting haplolines provide a foundation for off-the-shelf cell therapies in Spain and potentially across Europe, illustrating a scalable path toward broader ATMP accessibility while addressing immunological compatibility and regulatory requirements.