Kuebler et al. 2023 — Comprehensive notes on the clinical-grade HLA-homozygous iPSC haplobank for the Spanish population

Abstract and aims

• Goal: create a clinical-grade iPSC haplobank built from HLA-homozygous donors to cover a large fraction of the Spanish population, enabling off-the-shelf cell therapies with reduced immune rejection risk.

• Strategy: select cord blood units (CBUs) homozygous for the most common HLA-A, HLA-B, and HLA-DRB1 haplotypes; reprogram purified CD34+ cells to iPSCs; expand, bank under GMP; generate Master Cell Banks (MCBs) and Working Cell Banks (WCBs).

• Outcome: seven donors yielded seven haplotypes covering significant population fractions; GMP-compliant expansion and banking; first clinical-grade iPSC haplobank in Spain.

• Reprogramming method: Sendai virus-based CytoTune kit; transduction of CD34+ cells; virus-free status checked by RT-PCR; cells cultured in defined, xeno-free conditions.

• Applications: starting material for ATMP development and regenerative therapies; haplobank approach reduces time, cost, and need for patient-tailored lines.

Key concepts and definitions

• iPSCs: induced pluripotent stem cells derived from somatic cells that can differentiate into multiple lineages.

• Haplobank: a bank of iPSC lines homozygous for common HLA haplotypes to enable broad immunological matching across a population.

• HLA matching: focus on three loci for clinical relevance and population coverage: $ext{HLA-A}, ext{HLA-B}, ext{HLA-DRB1}$ with four-digit resolution.

• 0MM, 1MM, 2MM: zero-mismatch match (perfect across the three loci) and tolerances allowing 1 or 2 mismatches to estimate broader coverage.

• CD34+ cells: hematopoietic progenitors isolated from cord blood (CB) used as starting material for reprogramming.

• GMP: Good Manufacturing Practice; strict quality assurance framework for clinical-grade products.

• MCB/WCB: Master Cell Bank and Working Cell Bank, corresponding to scalable, traceable cell banks under GMP.

• ATMP: Advanced Therapy Medicinal Product; regulatory category for cell/gene therapies.

• Sendai virus (SeV): non-integrating reprogramming vector used to generate iPSCs; considered absent after verification.

• STR: Short Tandem Repeat profiling to confirm cell identity.

• QC assays: sterility, mycoplasma, endotoxin, adventitious virus testing; viability and recovery after thaw; karyotyping; pluripotency markers; EB differentiation.

Donor selection and population coverage

• Source: cord blood units from the Spanish Bone Marrow Donor Registry (REDMO).

• Donor process: reconsenting and ethics approval under IPS-PANIA project (ethics committee PR(AG)428/2018).

• Selection goal: homozygous CB samples for the most frequent HLA haplotypes to maximize coverage of the Spanish population.

• Coverage estimation: population cohort consists of combined REDMO adult bone marrow donors and CB donors; 4-digit HLA typing across 56,798 individuals.

• Coverage methodology:

  • Iterate haplotype by haplotype against the cohort; extract matched individuals; recompute remaining coverage for subsequent haplotypes.

  • Evaluate 0MM (no mismatches) and beneficial matches allowing 1MM or 2MM (one or two mismatches tolerated).

  • Cumulative coverage = (matched individuals per iteration / total sample) × 100.

• Reported coverage with 7 haplotypes (no mismatch): 21.37 ext{ } ext{%}; with 1MM: 50.83 ext{ } ext{%}; with 2MM: 92.46 ext{ } ext{%} (Fig. 1).

• Table 1 (haplotypes): seven haplotypes corresponding to iPSC lines named Hz 29-02/44:03/16:01/07:01/02:02/11:01 etc., including ABO type and donor sex (XY) and CD34+ counts prior to reprogramming.

Isolation and expansion of cord blood progenitors

• CBUs thawed, mononuclear fraction isolated by Ficoll in closed Sepax2 system.

• CD34+ selection:

  • Magnetic beads: CD34 MicroBead Kit; purification columns (MACS MS Columns).

• Expansion protocol (SP34 SFM+cytokines):

  • Medium: StemPro 34 Serum Free Medium + cytokines (50 ng/mL SCF, 50 ng/mL FLT3L, 10 ng/mL TPO, 10 ng/mL IL-6).

  • Duration: 4 days at 37°C, 5% CO₂; medium changed every other day.

• Post-expansion: cryopreservation of residual CD34+ cells for downstream steps.

Reprogramming CD34+ cells to iPSCs

• Reprogramming kit: CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit.

• Procedure:

  • Start with 1×10^4 CD34+ cells per transduction in SP34 SFM with cytokines and polybrene.

  • Overnight transduction at 37°C, 5% CO₂.

  • Remove residual Sendai virus; seed on Biolaminin CTG-coated dishes in SP34 SFM with cytokines.

  • Day 3–4: replace medium to SP34 SFM without cytokines; Day 6 onward: switch to Essential 8 Flex.

  • Monitor colonies daily for attached cell clusters indicative of reprogramming.

  • Emergence of colonies ~【16–18 days after transduction】; manually pick colonies and replate in Essential 8 Flex with RevitaCell on CTG laminin-coated dishes.

• Sendai clearance and validation:

  • After picking colonies, Sendai-infected cells are frozen for later testing.

  • RNA extracted; RT-PCR performed to confirm absence of Sendai virus genome; positive control included.

• Cloning and initial screening:

  • Karyotype analysis (G-banding) performed to confirm normal genome.

  • From clones, select one clone per line that is virus-free and karyotypically normal for further expansion.

Culture, cryopreservation, and clone selection

• Culture conditions:

  • Passaging with CTS DPBS and Versene; plating on CTG LN-521-coated plates; maintained in Essential E8 Flex.

• Clone selection criteria:

  • Absence of Sendai genome and transgenes by RT-PCR by passage ~7–8 with a 15-day 39°C culture period (to promote Sendai clearance).

  • Normal karyotype confirmed; one clone per line advanced for GMP expansion.

• Cryopreservation:

  • Clones thawed and expanded in GMP-like conditions; final banking in 40–50 vials with ≥1×10^6 viable cells per vial.

  • After freezing, data show viability and recovery metrics post-thaw.

GMP generation of MCB and WCB

• GMP expansion:

  • Clones thawed, expanded in classified facilities with A-grade hoods and B-grade background, using GMP-grade reagents.

  • Pre-banking testing for sterility, mycoplasma, and adventitious virus; viral testing performed prior to banking.

• Master Cell Bank (MCB):

  • Two vials of each validated homozygous clone thawed and expanded to create 6–8 dishes per clone to generate sufficient numbers.

  • Cells collected at appropriate confluence and cryopreserved in 40–50 cryovials; stored in liquid nitrogen after 24 h at −80°C.

• Working Cell Bank (WCB):

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

• QC focus during GMP phase:

  • Sterility, mycoplasma, endotoxin, adventitious virus status; viability by 7AAD; recovery and colony formation after thawing; total cell number per vial; STR identity; standard GMP documentation.

Characterization of iPSCs and banks

• Identity and pluripotency:

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

  • Alkaline phosphatase (AP) staining positive across lines.

• Differentiation potential:

  • Embryoid body (EB) formation in vitro; differentiation toward endoderm (AFP, FOXA2), ectoderm (TUJ1, GFAP), mesoderm (ASMA, GATA4).

  • Confocal imaging confirms lineage marker expression.

• Genetic identity and integrity:

  • STR profiling confirms identity; STRs match CD34+ CB cells (iPSCs derived from each donor).

  • Karyotype analysis performed at multiple stages (clone, banking); euploid status across lines: 3× 46,XX and 4× 46,XY.

• MCB/WCB characterization and QC:

  • MCB: full characterization including ICC for pluripotency, EB differentiation, STR; normal karyotype confirmed.

  • WCB: karyotype and STR checked; no major changes from MCB.

  • All MCB/WCB pass acceptance criteria for sterility, cell number, viability, and recovery post-thaw (Table 3).

Results: haplotype coverage and line properties

• Seven haplotypes established from CD34+ CB units, each homozygous for common Spanish haplotypes (Table 1).

• Haplotype details (examples):

  • iPS1-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; CD34 count Hz 30-18-3.

  • iPS2-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.

  • iPS3-Sv4F-E9, iPS4-Sv4F-F6, iPS6-Sv4F-H6, iPS7-Sv4F-I12, iPS8-Sv4F-J1 with corresponding haplotype strings (see Table 1).

• Coverage outcomes (Fig. 1): cumulative population coverage for the seven haplotypes under different match criteria:

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

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

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

• Clonal derivation and banking:

  • From each line, 10–14 clones were initially picked; 5 clones per line selected for expansion; by final selection, 1 clone per line advanced to full characterization and banking.

• Reprogramming efficiency and expansion (Table 2):

  • Expansion of CD34+ cells day1 and day4 (numbers in cells) and expansion factor; reprogramming efficiency per line ranged approximately from 0.45 ext{ } ext{%} to 1.8 ext{ } ext{%} depending on line.

  • Example data (per line):

  • Hz 29-44-7: day1 0.23×10^6; day4 0.87×10^6; expansion factor 3.87; reprogramming efficiency 0.86%

  • Hz 30-18-3: day1 1.14×10^6; day4 1.65×10^6; expansion factor 1.45; reprogramming efficiency 1.80%

  • Hz 3-7-15: day1 0.66×10^6; day4 1.17×10^6; expansion factor 1.77; reprogramming efficiency 1.07%

  • Hz 1-8-3: day1 2.20×10^6; day4 3.46×10^6; expansion factor 1.57; reprogramming efficiency 1.29%

  • Hz 33-14-1: day1 0.63×10^6; day4 1.57×10^6; expansion factor 2.49; reprogramming efficiency 0.45%

  • Hz 24-7-15: day1 1.37×10^6; day4 2.76×10^6; expansion factor 2.01; reprogramming efficiency 0.47%

  • Hz 11-27-1: day1 0.61×10^6; day4 1.056×10^6; expansion factor 1.73; reprogramming efficiency 1.34%

• Post-banking QC summary (Table 3): acceptance criteria include:

  • Sterility (Ph. Eur. 2.6.27): Negative

  • Mycoplasma (Ph. Eur. 2.6.7): Negative

  • Endotoxin (Ph. Eur. 2.6.14): <5 EU/mL

  • Adventitious viruses (cytopathic culture): Negative

  • Viability upon freezing (7AAD negative): >50%

  • Recovery 7 days after thawing: >20 colonies or 50% confluence

  • Total cell number before freezing: >1×10^6 cells/vial

  • 7AAD− viability: ≥50%

• Overall status: all MCBs and WCBs met quality control acceptance criteria and were registered in repositories (national stem cell bank and European registry).

Figures and tables referenced

• Fig. 1: Cumulative haplotype match coverage for the seven haplolines under 0MM, 1MM, and 2MM criteria.

• Table 1: Information on the seven established homozygous haplotypes and corresponding donor data (HLA-A, B, C, DRB1, DQB1, DPB1; ABO; sex; CD34; iPSC line name).

• Fig. 2: Morphology of CD34+ cells and generated iPSCs; ICC for pluripotency markers.

• Fig. 3: ICC with differentiation markers for EB-derived cells (endoderm, ectoderm, mesoderm).

• Table 3: QC acceptance criteria for GMP-MCB and GMP-WCB.

• Supplementary: Additional Fig. S1 shows absence of Sendai virus signals by PCR; Table S2 STR details; Fig. S2 karyotype; Fig. S3 AP activity; Fig. S1, S2, S3 in Additional file 1.

Discussion: context and comparisons to other populations

• Population coverage context:

  • Compared with other haplobank efforts: Japanese population estimates indicate 30 lines for ~82.2% coverage at three-locus concordance; 140 lines can reach ~90% coverage for Japanese; 50 lines could cover 90% for European populations in some estimates depending on haplotype distribution.

  • In Korea, 22 GMP-compliant homozygous HLA-type iPSC lines cover about 51% of the population; 13 lines in Korea (GMP) from blood and CB for 50% coverage in that population.

  • European and other populations require fewer lines with haplotype sharing, but initial banking must reflect regional haplotype frequencies.

• Practical implications:

  • Haplobanks reduce time and cost for patient-specific iPSC therapy; potentially lower need for immunosuppression if HLA-matched.

  • For some applications, looser matching (allowing 1–2 mismatches) provides broader coverage (e.g., 50%–> nearly entire Spanish population with seven haplolines).

  • Cord blood banks offer several advantages: healthy, young donor cells with fewer somatic mutations; CBUs are already HLA-typed; established clinical manufacturing frameworks exist for CB-derived lines.

• Regulatory and ethical considerations:

  • iPSC derivatives for cell therapy are regulated as ATMPs; GMP-compliant manufacturing and strict QA are required.

  • The project emphasizes traceability, donor consent (IPS-PANIA), and public accessibility of haplobank data via registries (hPSCReg).

  • There is ongoing discussion about comparability across lines and facilities, standardization of QC attributes, and ISSCR guidelines for stem cell use in research.

Ethical, regulatory, and practical implications

• Ethics:

  • Informed consent and donor re-contacting; withdrawal rights; ethical oversight via CEIC approvals; transparency about donor data usage.

  • Privacy and data protection in correlating donor HLA haplotypes with population coverage and clinical use.

• Regulatory:

  • Banking and expansion occurred under GMP conditions; final products are intended as intermediate materials for ATMP development rather than direct cell therapy products.

  • Compliance with EMA and FDA guidance for hPSC derivatives; use of xeno-free and clinically approved reagents; validated by quality control laboratories.

• Practical implications for clinicians and industry:

  • Haplobank lines can act as starting materials to generate multiple differentiated cell products for diverse indications (ophthalmologic, hematologic, cardiac, neurological, metabolic diseases).

  • A global European registry (hPSCReg) and COST network support standardization, data sharing, and educational programs to enable broader adoption.

Conclusions

• The haplobank described represents a valuable intermediate cell bank covering 21.37 ext{ } ext{%} of the Spanish population at strict multi-locus matching (0MM) and even broader coverage when allowing mismatches (50.83 ext{ } ext{%} for 1MM and 92.46 ext{ } ext{%} for 2MM).

• The seven haplotypes share haplotype blocks common to broader European and American populations, enabling international utility beyond Spain.

• This work lays a foundation for national haplobanks and international sharing of haplotype-matched iPSCs, enabling safer, faster production of derivatives for diverse diseases.

• The combination of CB-derived starting material, Sendai-based reprogramming, GMP expansion, and multi-layer QC demonstrates a rigorous path toward clinically relevant iPSC banks while highlighting the need for ongoing standardization and regulatory alignment.

Abbreviations (selected)

• ATMP: Advanced Therapy Medicinal Product

• BAM: not used here
  -CB: Cord Blood

• CBU: Cord Blood Unit

• CRS: not used here

• CST: not used here

• DPS: not used here

• EB: Embryoid Body

• EMA: European Medicines Agency

• FDA: Food and Drug Administration

• GMP: Good Manufacturing Practice

• hPSC: human Pluripotent Stem Cells

• HI: not used here

• HLA: Human Leukocyte Antigen

• iPSC: Induced Pluripotent Stem Cell

• MCB: Master Cell Bank

• MOI: multiplicity of infection

• QCs: quality controls

• PCB: not used here

• REDMO: Spanish Bone Marrow Donor Registry

• RPM: not used here

• SFM: Serum-Free Medium

• SeV: Sendai Virus

• STR: Short Tandem Repeats

• WCB: Working Cell Bank

Notes on data accessibility and supplementary information

• All presented data are available for consultation; supplementary information includes detailed QC data, additional figures showing Sendai clearance, and extended tables of STRs and haplotype details.

• The online supplementary materials provide deeper insight into reprogramming conditions, colony selection, and QC assays (Additional file 1).

Quick reference to key numerical data (LaTeX-ready)

• Coverage (0MM): $21.37\%$

• Coverage (1MM): $50.83\%$

• Coverage (2MM): $92.46\%$

• Sample size for population coverage: $56{,}798$ individuals

• Reprogramming efficiency range across haplotypes: $0.45\%\text{ to }1.80\%$

• Expansion metrics (example line Hz 30-18-3): day1 $1.14\times 10^6$ cells; day4 $1.65\times 10^6$ cells; expansion factor $1.45$

• Viability criteria: 7AAD−, >50% viability upon freezing; recovery >20 colonies or >50% confluence after thawing

• Karyotype status: all lines euploid; distribution: $3\times 46,XX\quad +\;4\times 46,XY$

References to figures and tables (contextual)

• Fig. 1: cumulative haplotype coverage by match stringency

• Table 1: seven haplotype entries and donor data

• Table 2: CD34+ expansion and reprogramming efficiency per line

• Table 3: QC acceptance criteria for GMP-MCB and GMP-WCB

• Fig. 2: morphology and pluripotency marker ICC

• Fig. 3: EB differentiation markers

• Supplementary Figures S1–S3 and Tables S1–S3 provide validation and extended QC data