Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 https://doi.org/10.1186/s13287-022-02961-6 RESEARCH Open Access Public Cord Blood Banks as a source of starting material for clinical grade HLA‑homozygous induced pluripotent stem cells Belén Álvarez‑Palomo1,2* , Anna Veiga3, Angel Raya3, Margarita Codinach1,4, Silvia Torrents1, Laura Ponce Verdugo5, Clara Rodriguez‑Aierbe6,7, Leopoldo Cuellar8, Raquel Alenda9, Cristina Arbona10,11, Dolores Hernández‑Maraver12, Cristina Fusté13 and Sergi Querol1,2 Abstract Background: The increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP- compliant cell isolation process. Methods: Homozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns. Results: We obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population. Conclusions: Here we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy. Keywords: Hematopoietic progenitor cells, Cord blood, Cord blood banks, Induced pluripotent stem cells, HLA matching, GMP manufacturing Background Induced pluripotent stem cells (iPSC) can be generated in the laboratory from donor adult somatic cells by the reprogramming process, displaying similar characteris- tics to embryonic stem cells and free of the ethical con- *Correspondence: abalvarez@bst.cat cerns related to embryo use [1]. IPSC can be expanded 1 Cell Therapy Service, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, unlimitedly and they can be differentiated into any cell Passeig de Taulat, 106‑116, 08005 Barcelona, Spain Full list of author information is available at the end of the article type in the body; moreover, the genetic background of the © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 2 of 11 donor can be chosen. These features make iPSC excep- The most common strategies to obtain haplolines from tional candidates as starting material for cell therapies blood samples consists in isolating the mononuclear frac- [2]. IPSC-derived cells are being tested at the moment in tion or the CD34+ hematopoietic progenitor population clinical trials for regenerative medicine to treat diseases [21, 22]. such as age-related macular degeneration [3] or Parkin- In this article, we report our experience to obtain son’s disease [4], and for immunotherapies for conditions CD34+ from homozygous donors to make iPSC haplo- such as cancer [5] or graft-versus-host disease [6]. lines following a procedure that complies with regulatory Although the production of therapeutic cells from the standards for ATMP starting materials and that can be patient’s own iPSC would provide no graft rejection due adopted by other CBB with an interest in creating iPSC to histocompatibility issues, the high cost and the long haplobanks. The process was performed within the Span- duration of iPSC production plus differentiation to the ish haplobank project (IPS-PANIA) on nine CB units therapeutic cell aroused considerations over the practi- corresponding to HLA haplotypes estimated to provide cality of an autologous use of iPSC. Several approaches the widest possible coverage of the Spanish population for the allogenic use of iPSC have been proposed, like [12]. genome editing to eliminate HLA expression [7] or HLA- matching, similarly to what is done for organ or bone Materials and methods marrow transplantation [8]. It was proposed that creating Safety control of CBU accepted in the bank iPSC lines from HLA homozygous donors would provide All the CBU candidate for being included in a CBB are a match for a wide percentage of the population with a tested for microbial sterility by colorimetric culture assay relatively small number of iPSC lines [9]. This approach and for the presence of antibodies against common blood has shown promising results in primates [10]. Since HLA borne viruses: hepatitis B (HBV), VIH 1/2, hepatitis C haplotypes differ in each geographical area, several stud- (HCV), HTLV-I/II, cytomegalovirus and Epstein Barr ies have identified the most common haplotypes that and for the presence of VHB DNA and HVC and VIH-1 would serve a national or regional population and calcu- RNA. The presence of antibodies against parasites Toxo- lated the number of HLA homozygous iPSC lines (haplo- plasma gondii, Trypanosoma cruzi and Treponema pal- lines) to cover a significant percentage [9, 11, 12]. Cord lidum are also assessed. blood banks provide an excellent source of starting mate- rial for the production of iPSC: the samples are banked in Segment verification great numbers, they are already HLA-typed and screened High-resolution typing was performed by Sanger for the presence of virus and pathologies [13]. Moreover, sequencing in an ABI PRISM 3130xl Genetic Analyzer CB cells are young cells with negligible risk of accumu- (ThermoFisher) and/or next generation sequencing lated genetic and epigenetic insults and easy to repro- (NGS) in a MiSeq platform (Illumina) or in Ion GeneStu- gram. Several countries have set up initiatives to create dio S5 System (ThermoFisher) for HLA-A, -B, -C, -DRB1, their own banks of haplolines (haplobanks) using banked -DRB3/4/5, -DQB1 and -DPB1 genes. CD45+ and CB as a source of starting cells, with Japan and Korea CD34+ cell content was assessed by flow cytometry fol- reporting already the first haplolines [14, 15]. lowing ISHAGE protocol and viability was calculated by In order to use iPSCs in cell therapy, haplolines must dye exclusion with 7AAD. Colony formation assays to be produced to clinical grade, complying with GMP calculate the expected clonogenic efficiency (E-clone) standards throughout manufacturing [16]. Several inter- was done in StemMACS HSC-CFU methyl-cellulose national efforts have been put in place to create a set of medium (Miltenyi) and cultivation for 14 days in a ­CO2 standards for critical quality attributes of iPSC for clini- cell 37 °C, 5.5% CO2, 90% HR incubator. cal applications, seeking the international harmonization to allow for haplolines exchange among haplobanks and a Thawing of the CBU and mononuclear fraction (MNF) better coverage of the population [17, 18]. purification Several crucial aspects have to be considered in the The CB bags were thawed in a water bath at 37 °C and production of clinical grade iPSC, starting with the pro- slowly equilibrated with a 1:1 volume of equilibra- curement of the starting cells. Banked CB provides a tion buffer containing PBS with no Ca2+ or Mg2+, 5% source of cells that have been already procured follow- p/v human serum albumin (Griffols), 5 mM M ­ gCl2 and ing clinically regulated standards such as Jacie [19] or 200U/mL of DNAse I (Pulmozyme, Roche) in a closed Netcord [20], that assure the compliance with therapeu- system and allowed to equilibrate at 4 °C for 5 min. The tic use standards; however, their use for reprogramming mononuclear (MN) fraction was purified in a closed requires signing a new informed consent explaining the system using ficoll (Histopaque-1077, Sigma-Aldrich) novel applications of the donation. in a Sepax-2 cell processor (Biospace) with the NeatCell Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 3 of 11 program and using the CS900.2 kit. PBS with no Ca2+ or and the Informed Consent document, and they were Mg2+, 2% p/v human serum albumin (Griffols), 2.5 mM invited to read them carefully; both documents had been ­MgCl2 and 20U/mL of DNAse I (Pulmozyme, Roche) was approved by the Vall d’Hebron Hospital Ethics Commit- used as washing buffer. The obtained MNF was diluted tee for Human Research. The donors that were interested 1:1 with washing buffer then centrifuged for 15 min at in participating had an interview, either in person or 300 g. The supernatant was discharged and the pellet by telephone, with a physician who knew the project in resuspended at a concentration of 2 × ­106 MN cells/mL detail but was not directly involved in it. Those donors in washing buffer. consenting on participating also answered a health ques- tionnaire to discard genetic diseases or other incompat- CD34+ selection ibilities with the donation (Additional file 1: Fig. S1.). The cell suspension was blocked for 5 min at 4 °C with All the selected donors were reachable by phone and 100 ul/108 cells of Planngamma 100 mg/mL (Griffols). willing to listen to the proposal. An in-person interview CliniMACS CD34 reagent (Miltenyi) immunomagnet- was the first choice to meet the donor and to give them ics beads were added at 100 ul/108 cells and incubated a chance to ask questions about the donation and the for 30 min at room temperature, mixing every 5 min. project, and to sign the Informed Consent document. Subsequently the cells were washed in 50 mL of wash- However, due the COVID-19 pandemic, for most of the ing buffer and centrifuged 15 min at 300 g. The labeled donors the interviews were performed over the phone cells were then passed through a clinical grade separation and the Informed Consent was sent by courier to the mini column (MACS ART MS columns Miltenyi) located donor’s home and collected on the spot. ­ 08 cells two consecu- in a fixed magnetic field. For each 1 From the 17 donors recontacted, only three of them tive columns were used. CD45+ and CD34+ content and declined to participate and one of them was discarded viability were assessed right after purification and after due to a chromosomal abnormality found in a post-dona- o/n incubation by flow cytometry using anti-CD45 (clone tion miscarriage. J33, Beckman), anti-CD34 (clone 581, Beckman) and 7AAD (Beckman) for viability. CBU selection criteria The whole Spanish cord blood registry comprises 52,220 CD34+ expansion donations. From the 9419 which are genotyped in high CD34+ selected cells were resuspended and cultivated resolution, we identified 109 homozygous units corre- in Stempro34 (Gibco) supplemented with IL6 10 ng/mL, sponding to 43 different haplotypes and we ranked the IL3 10 ng/mL, TPO 10 ng/mL, SCF 50 ng/mL and Flt3l haplotypes priority according to the cumulative coverage 50 ng/mL. All cytokines were human recombinant GMP capacity for HLA matching, as previously described [12]. grade (PeproTech). The average pre-freeze content in CD34+ in the homozy- gous units was 5.51 × ­106. The screening selection criteria Sterility and mycoplasma testing for the homozygous CBU are listed in Table 1. Sterility was assessed by BactAlert IFA and iFN plus bot- As an initial selection criterion, only those haplotypes MycoAlert™ Detection Kit (Lonza). tles (Biomerieux) and the presence of mycoplasma by represented in more than two homozygous cord blood units were considered, in order to keep always at least one unit of the haplotype available for transplantation Results within any of the national Cord Blood Banks. A second An overview of the process we followed to obtain clini- selection criteria was to have a pre-freeze CD34+ content cally compatible homozygous CD34+ is depicted in of ≥ 2 × ­106 cells. Selected units were pre-reserved from Fig. 1, with critical steps and go/no go points. the Spanish CB registry. Verifications on frozen segments were performed for 25 units with the goal to create seven Donor recontact and reconsent haplolines representing the top haplotypes estimated to All cord blood donors—the mothers of the new-borns give the widest coverage for the Spanish population. Ini- whose umbilical cord blood is collected—recontacted tially, we set as acceptance criteria having an E-clone of had agreed in the informed consent for the original at least 10%, as a measure of hematopoietic progenitor cord blood donation on the possibility of being con- potency, and a CD34+ viability after thawing of at least tacted again in the future. The donors were telephoned 50%. Since segments normally perform worse than the and informed about the project and the special interest main bag in terms of viability upon freezing, some sam- of their homozygous donated samples. Subsequently, ples with low performance on CD34+ viability or colony an e-mail was sent including an introductory brochure formation at the verification, were still considered as can- on simple language, the Donor Information document didates taking into account other considerations such as Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 4 of 11 Fig. 1 Process diagram for homozygous CB units (CBU) selection and processing. IC = Informed Consent Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 5 of 11 Table 1 Homozygous CB units initial selection criteria 98%. Upon thawing, the median viability was still very high, 90.5% [50.9–99.3%], and the median recovery of Available CBU in Spanish banks ≥2 CD34+ was 67.6% [17.5–88.1%]. The time of storage in CD34+ count pre-freeze [10E+06] ≥2 cryopreservation, ranging from 5.1 to 22.8 years, did not Post-thaw viability CD34+ (%) ≥ 50 seem to affect the viability or the recovery (Fig. 3). E-clone (%) > 10 For CD34+ selection we used the clinical grade immu- ABO type 0 nolabelled beads from Miltenyi (CliniMACS CD34 rea- gent) but not the CliniMACS device, since the device, column and tubing system are designed for larger vol- umes and much higher content of CD34+ (up to 6 × ­108). CD34+ content at collection or the quality of alternative The CD34+ purification was thus performed in an open candidate CBU for that haplotype. On the thawed seg- system in an A grade hood within a D grade environ- ments, the median viability of CD45 + cells was 51.4% ment. A clinical grade separation column placed in a [15.9–88%] and that of CD34+ was 82% [36.8–98.7%] and fixed strong magnetic field was used to select the labelled the median E-clone was 24.6% [0.6–130.9%] (Table 2). cells. To improve the purity of the final population, the As part of the verification process, the samples HLA cells passed through two selection columns sequentially. was retyped by NGS to confirm homozygosity. Finally, Figure 3 shows the qualitative analysis during CD34+ iso- nine units were thawed for CD34+ selection, prior lation. One of the CB units (process n. 2) did not perform reserve confirmation to the Spanish CB registry. The to the required standards and the process did not pro- characteristics of the selected CBU are described in ceed after column purification. We suspect this was due Table 3. to the quality of the freezing process and not the thawing and isolation, since the quality standards and NC yield Thawing of CBU and CD34+ selection were low since the first steps of the process. For the rest We designed a thawing and CD34+ cell isolation pro- of the samples, after column purification we obtained a cess that could be consistent and that yielded a sufficient median of 1.39 × ­106 total CD34+ [0.94 × ­106–2.98 × ­106] number of viable CD34+ cells for reprogramming and set cells, resulting in a median recovery of 20% [10.6–84%] the acceptance criteria for the final CD34+ purified pop- of the collected CD34+ and a 34.1% [15.1–56.8%] of the ulation, represented in Table 4. content at thawing. The median viability at this point was The whole thawing, mononuclear fraction (MNF) 99.1% [96.7–99.5%] and the purity 83.5% [66.9–87.9%]. enrichment and CD34+ purification process was per- After column selection the cells were incubated o/n in formed in the clean room facilities—in an A grade hood CD34+ expansion medium and quality tested again. within a D grade environment—and using GMP grade The average total number of CD34+ slightly declined reagents compatible with clinical use. Each CBU was o/n (median 1.05 × ­106 CD34+ [0.53 × ­106–3.73 × ­106]) retrieved from liquid nitrogen storage and code veri- but viability and purity remain quite constant (medians fied before transporting it in a dry shipper to the clean 96.1% [76.3–98%] and 83.5% [66.9–88.3%], respectively). room. Upon the point of MNF purification, the proce- Samples were taken for sterility testing after o/n incu- dure was performed in a closed system. The CBU thawed bation and for mycoplasma testing after 72 h in culture, at 37 °C in a water bath were slowly equilibrated with a giving a negative result in all cases. In all cases, the puri- 5% p/v human serum and DNAse I containing solution fied CD34+ population was allowed to expand for 4 days and a sample was retrieved for a cytometry assay. At this before reprogramming. After an initial drop in cell num- point the median content on nucleated cells (NC) was ber, by day 4 the CD34+ cells had recovered and were 1.77 × ­109 [5.41 × ­108–2 × ­109] cells, meaning a median proliferating again. The median expansion fold at this recovery 94.7% [63.4–111.3], with a median viability point was 1.75 [0.96–3.87]. 1 × ­104 CD34+ were used for of CD45+ of 48% [45.6–69.4%] (Fig. 2). Isolation of the reprogramming and the rest were kept as a backup and as MNF with the ficoll method was performed in a Sepax control for future quality control assays. 2 cell processor, using the NeatCell program, directly from equilibration without prior washing. This method allowed for a closed system and consistent recovery of Discussion the MNF with ficoll. At this point, the NC content was Since the number of potential clinical applications for reduced to a median 4.3 × ­108 [2 × ­108–8.3 × ­108] which iPSC-derived cells increases and clinical trials progress, correspond to a 25.3% [3.7–48.9%] of recovery (Fig. 2). the need for clinical grade quality iPSC becomes more The range of CD34+ content of the CBU pre-freeze evident. The use of banked CB units as a source of HLA ranged from 2 to 17.9 1 ­ 06 cells and viability was a median homozygous donor cells to create allogenic iPSC cell therapies has become a sensible and attractive option Table 2 Quality control data from CB units segments verifications Haplotype Rank cover CBU Freeze date Pre-freeze Post-thaw SEX ABO NC ×10e9 CD34+ ×10e6 CD45+ 7AAD- % CD34+ 7AAD- % Eclone % 29:01–44:03–07:01 1 1 04/11/2010 2.68 8.7 15.9 36.8 3.6 M O POS 2 11/20/2009 2.41 11.8 34.8 62.4 28.0 M O POS 3 05/17/2013 2.19 7.9 47.0 81.0 19.6 F O NEG 4 10–11-2008 0.93 2.8 53.1 70.0 75.9 F O NEG 01:01–08:01–03:01 2 1 12–06-2009 1.63 17.9 76.8 89.0 20.4 F O NEG 2 10/27/2009 2.23 6.8 36.7 49.6 11.0 F A POS Álvarez‑Palomo et al. Stem Cell Research & Therapy 3 10/30/2008 0.95 8.29 88.0 90.0 7.2 F O POS 30:02–18:01–03:01 3 1 05/23/2015 2.10 7.2 90.4 15.9 M O POS 2 12/20/2004 1.67 6.8 31.0 66.0 0.56 F O POS 3 03–04-2009 2.18 5.2 56.9 73.0 71.1 F O NEG 03:01–07:02–15:01 4 1 04/10/2007 1.88 11.4 77.2 91.0 46.6 M O POS (2022) 13:408 2 02/25/2003 2.40 5.5 50.0 86.0 8.2 M B POS 3 06/16/2008 2.05 12.6 45.0 65.0 27.0 F A POS 02:01–44:03–07:01 5 1 03/04/2008 1.06 2.9 61.2 82.0 41.1 M O POS 2 03/02/2011 1.17 2.3 70.0 70.0 130.9 M A POS 02:01–07:02–15:01 6 1 05/15/2009 1.55 9.3 81.3 86.0 12.1 F B POS 33:01–14:02–01:02 7 1 02/12/1999 0.96 5.1 50.0 78.0 13.9 F O NEG 2 03/17/2003 1.26 2.7 45.0 98.7 49.8 M O POS 02:01–51:01–11:01 8 1 04/14/2018 2.30 2.7 85.5 F B POS 2 11/14/2006 1.70 6.7 51.0 91.0 21.0 F O POS 01:01–57:01–07:01 10 1 03/21/2008 2.17 2.8 51.7 86.6 36.0 F O NEG 2 09/27/2001 0.89 1.2 62.0 91.0 41.2 F A NEG 24:02–07:02–15:02 1 02/27/2016 1.86 9.0 42.4 71.8 34.0 M O POS 11:01–27:05–01:01 1 02/05/2015 1.47 5.2 35.9 58.3 34.5 F O POS 2 10/14/2003 1.10 3.4 82.1 95.0 25.9 F A POS 11:01–35:01–14:54 1 09/21/2009 1.00 4.2 86.6 94.0 18.6 F O POS Median 1.63 6.1 51.4 82.0 24.6 Range 0.89–2.68 1.2–17.9 15.9–88 36.8–98.7 0.6–130.9 Several units were verified for each of the haplotypes that provided higher HLA matching coverage on the Spanish population (Rank cover.). NC = nucleated cells. Lines in bold correspond to the CB units finally selected for thawing Page 6 of 11 Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 7 of 11 Table 3 Selected CB units characteristics: High resolution HLA typing, sex, ABO type, volume of CB on collection and days in storage from collection to thawing CBU Freq. haplotype HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQB1 HLA-DPB1 ABO type Sex CB volume Days in storage collected (mL) 1 0.0311 29:02 44:03 16:01 07:01 02:02 11:01 O+ XY 141.5 3709 2 0.0262 01:01 08:01 07:01 03:01 02:01 03:01/04:01 O+ XX 88 4459 3 0.0262 01:01 08:01 07:01 03:01 02:01 01:01/03:01 O− XX 134 4078 4 0.0196 30:02 18:01 05:01 03:01 02:01 02:02/04:01 O+ XY 115.5 1958 5 0.0135 03:01 07:02 07:02 15:01 06:02 02:01/04:01 O+ XY 130 5035 6 0.0083 02:01 44:03 16:01 07:01 02:01 11:01/19:01 O+ XY 78 4748 7 0.0096 33:01 14:02 08:02 01:02 06:02 02:01P O− XX 123 8339 8 0.0052 24:02 07:02 07:02 15:01 06:02 04:01P O+ XY 126.5 1864 9 0.0040 11:01 27:05 01:02 01:01 05:01 04:01/11:01 O+ XX 105.5 2338 as well as a way to revalorize existing CBB. Contrary to donations and the samples are already screened for medi- peripheral blood or other tissues as starting material cal fitness and tested for viral infections and sterility. for reprogramming, banked CB does not require new Japan and Korea have reported the production of the first sets of haplolines for their national haplobanks and Fig. 2 Nucleated cells (NC) recovery during the process. A Total number of NC measured in automatic cell counter. B Recovery or % vs the initially reported NC content upon collection. C CD45 + viability measured by dye exclusion at the cytometer, before and after freezing. Lines depict median values Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 8 of 11 Fig. 3 CD34+ population recovery during the process. A Total number of CD34+ measured by flow cytometry. B Recovery or % vs the initially reported CD34+ content upon collection. C CD34+ viability measured by dye exclusion at the cytometer, at different stages of the process. D Purity calculated as % of viable CD34+ within the CD45+ viable population of the immunoselected cells just after the purification column and after o/n incubation. E Purified CD34+ population expansion after 96 h in culture. Lines depict median values other countries, such as Germany, Spain or Australia, and safeguard the potential for hematopoietic progenitor have initiated the construction of theirs. The IPS-PANIA transplantation, only those haplotypes that were repre- project aims at the production of seven clinical grade sented in at least two homozygous CB units we consid- haplolines representing top haplotypes regarding HLA- ered. This did not affect the top four most common match coverage of the Spanish population. Not to deploy haplotypes but for some of the less common haplotypes the Spanish CB registry of any particular HLA haplotype we could not find sufficient homozygous CB units. Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 9 of 11 Table 4 Acceptance criteria for the immunoselected haplobank in Germany using a CliniMACs device. Con- CD34+ purified population to continue into reprogramming sidering the small scale in volume and CD34+ content process of cryopreserved CBU we decided not to use the Clini- CD34+ count [10E+05] ≥1 MACS device, which is designed for larger samples and CD34+ viability (7AAD neg %) ≥ 80 up to tow orders of magnitude higher content of CD34+. CD34 purity (%)* ≥ 70 We also considered the high cost in reagents and con- Sterility Negative sumables of using the ClinicMACS methods for cryo- preserved CB samples. Alternatively, we used a scaled Mycoplasma Negative amount of the CliniMACS CD34 reagent and clinical *Purity calculated as % of viable CD34+ within the CD45+ viable population grade columns, and performed a manual immunoselec- tion. Using the described method, we obtained good quality CD34+, with a good recovery, which proliferated Since the original CB donation had a specific aim for and reprogrammed satisfactorily—only one of the eight hematopoietic progenitor transplantation and the par- thawed CBU failed to reprogram. ticular nature of iPSC cells—unlimited expansion and ever-growing possibilities of applications and potentially Conclusions multiple patients—the recontacting for the signing of a We report our own experience in producing HLA- new Informed Consent document deserves special care. homozygous CD34+ from cryopreserved CBU as star- The donors have to be clearly informed of the new nature ing material for clinical grade iPSC and we propose an of the donation and the wide possibilities of the samples. efficient and reproducible protocol, from donor recon- In our experience, contacting the donors by telephone tacting to a cost-effective and clinical grade-compliant by qualified personnel not directly involved in the pro- CD34+ isolation and expansion procedure. Our aim is ject was an efficient way to track back the donors and a to contribute to the production of internationally har- favorable predisposition to participate was high. Adapt- monized haplolines as sources of cell products for clini- ing to the COVID-19 pandemic restrictions and switch- cal research and therapy. ing from in-person interview to phone calls was well accepted. Most of the recontacted donors (82%) agreed to the new use of the donation. Abbreviations We set a series of requirements on the homozygous CB: Cord blood; CBB: Cord blood bank; CBU: Cord blood unit; E-clone: Expected clonogenic efficiency; iPSC: Induced pluripotent stem cells; NC: CBU to be selected for consideration, regarding firstly Nucleated cells. cellularity and CD34+ content, to assure a sufficient number of cells to reprogram and for backup storage Supplementary Information after purification and recovery. Although having an O The online version contains supplementary material available at https://​doi.​ group was initially considered preferential but no an org/​10.​1186/​s13287-​022-​02961-6. essential requirement, as ABO blood group incompatibil- ity has been described as a possible risk of graft rejection Additional file 1: Fig. 1. Health questionnaire carried out on cord blood donors once they agree to participate in the study. [23], and we found availability of O type donors to for the haplotypes providing the widest coverage, we decided to include it in the requirements list. Similarly, a minimum Acknowledgements We would like to acknowledge Mrs. Elena Valdivia for technical advice, Dr. requirement of homozygosity in HLA-A, HLA-B and Jesús Fernandez and Dr. Laura Medina for contacting the donors and Mrs. HLA-DRB1 was originally set, but preference was given Elisabet Tahull Navarro for administrative and management help. We would to CBU that were also homozygous for HLA-C. Indeed, also like to thank the cord blood donors, the donation programs professionals, and all the cord blood banks staff for their invaluable dedication to helping all the selected units chosen for reprogramming in the bring hope to many patients in need. IPS-PNIA project are also homozygous for the HLA-C allele. Rhesus type and sex were not considered as selec- Author contributions BAP, has participated in the design of the work; the acquisition, analysis and tion requirements as there is no evidence that there could interpretation of data; and draft of the document. SQ has participated in the a significant impact in the future engraftment of the design of the work, analysis, interpretation of data and draft of the document. iPSC-derived cells. AV and AR, have participated in the design of the work, analysis and interpre‑ tation of the data and revision of the document. MC, ST, LPC, CRA, LC, RA, CA, To thaw and enrich the mononuclear fraction we DHM and CF have participated in the acquisition of data and revision of the decided to use the Sepax cell processor, without prior document. All authors read and approved the final manuscript. washing, similarly as it was described by Kaur and col- Funding leagues [24]. For the CD34+ purification, Liektde and This research was funded by the Spanish Ministry of Science, Innovation and colleagues [19, 21] have reported satisfactory results Universities, National Plan for Scientific and Technical Research and Innovation processing HLA-homozygous CBU for the creation of a Álvarez‑Palomo et al. Stem Cell Research & Therapy (2022) 13:408 Page 10 of 11 2013–2016 RETOS COLABORACIÓN Program 2017: exp. RTC-2017-6000-1. dose-escalation study. Nat Med. 2020;26(11):1720–5. https://​doi.​org/​10.​ 2017–2021. 1038/​s41591-​020-​1050-x. 7. Lee J, et al. Abrogation of HLA surface expression using CRISPR/ Availability of data and materials Cas9 genome editing: a step toward universal T cell therapy. Sci Rep. All the presented data is available for consultation. 2020;10(1):17753. 8. Taylor CJ, Bolton EM, Pocock S, Sharples LD, Pedersen RA, Bradley JA. Banking on human embryonic stem cells: estimating the number of Declarations donor cell lines needed for HLA matching. Lancet. 2005;366:2019–25. 9. Pappas DJ, Gourraud PA, Le Gall C, Laurent J, Trounson A, DeWitt N, Talib Ethics approval and consent to participate S. Proceedings: human leukocyte antigen haplo-homozygous induced The Information Document and the Informed Consent form sent to the pluripotent stem cell haplobank modeled after the California popula‑ donors were approved by the Ethics Committee for Research with Medicines tion: evaluating matching in a multiethnic and admixed population. from Vall d’Hebron Hospital (Barcelona, Spain). All donors participating had Stem Cells Transl Med. 2015;4(5):413–8. https://​doi.​org/​10.​5966/​sctm.​ signed the Informed Consent document. 2015-​0052. 10. Sugita S, et al. Successful transplantation of retinal pigment epithelial Consent for publication cells from MHC homozygote iPSCs in MHC-matched models. Stem Cell Not applicable. Rep. 2016;7(4):635–48. 11. Taylor CJ, Peacock S, Chaudhry AN, et al. Generating an iPSC bank for Competing interests HLA-matched tissue transplantation based on known donor and recipi‑ The authors declare no competing interests. ent HLA types. Cell Stem Cell. 2012;11(2):147–52. https://​doi.​org/​10.​ 1016/j.​stem.​2012.​07.​014. Author details 12. Álvarez-Palomo B, García-Martinez I, Gayoso J, Raya A, Veiga A, Abad ML, 1 Cell Therapy Service, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Eiras A, Guzmán-Fulgencio M, Luis-Hidalgo M, Eguizabal C, Santos S, Passeig de Taulat, 106‑116, 08005 Barcelona, Spain. 2 Transfusional Medicine Balas A, Alenda R, Sanchez-Gordo F, Verdugo LP, Villa J, Carreras E, Vidal Group, Vall d’Hebron Research Institute, Autonomous University of Barce‑ F, Madrigal A, Herrero MJ, Rudilla F, Querol S. Evaluation of the Spanish lona (UAB), Barcelona, Spain. 3 Programa de Medicina Regenerativa, Institut population coverage of a prospective HLA haplobank of induced pluripo‑ d’Investigació Biomèdica de Bellvitge. IDIBELL, Hospital Duran i Reynals, tent stem cells. Stem Cell Res Ther. 2021;12(1):233. https://​doi.​org/​10.​ Gran Via de L’Hospitalet, 199‑203, 08908 L’Hospitalet de Llobregat, Barcelona, 1186/​s13287-​021-​02301-0. 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