Cytotherapy 26 (2024) 334 339

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CYTOTHERAPY journal homepage: www.isct-cytotherapy.org

FULL-LENGTH ARTICLE
Immunotherapy

Source of hematopoietic progenitor cells determines their capacity to

generate innate lymphoid cells ex vivo
Said Z. Omar1,2, Vera van Hoeven1,2, Nienke J.E. Haverkate1,2, Jolien M.R. Van der Meer2,3,
Carlijn Voermans2,3, Bianca Blom1,2, Mette D. Hazenberg1,2,3,4,5,*
1
Department of Experimental Immunology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
2
Amsterdam Infection and Immunity Institute, Amsterdam, The Netherlands
3
Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
4
Cancer Center Amsterdam, Amsterdam, The Netherlands
5
Department of Hematology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands

A R T I C L E I N F O A B S T R A C T

Article History: Background aims: The success of allogeneic hematopoietic cell transplantation (HCT) as therapy for hemato-
Received 17 July 2023 logic conditions is negatively impacted by the occurrence of graft-versus-host disease (GVHD). Tissue dam-
Accepted 30 January 2024 age, caused, for example, by chemotherapy and radiotherapy, is a key factor in GVHD pathogenesis. Innate
lymphoid cells (ILCs) are important mediators of tissue repair and homeostasis. The presence of ILCs before,
Key Words: and enhanced ILC reconstitution after, allogeneic HCT is associated with a reduced risk to develop mucositis
allogeneic HCT and GVHD. However, ILC reconstitution after allogeneic HCT is slow and often incomplete. A way to replenish
GVHD the pool of ILC relies on the differentiation of hematopoietic progenitor cells (HPCs) into ILC.
immune reconstitution
Methods: We developed an ex vivo stromal cell containing culture system to study the capacity of HPCs to
ILC
differentiate into all mature helper ILC subsets.
ILC development
Results: ILC development depended on the source of HPCs. ILCs developed at high frequencies from umbilical
cord blood and fetal liver derived HPC and at low frequencies when HPCs were obtained from allogeneic
or autologous adult HCT grafts or healthy adult bone marrow. Although all helper ILC subsets could be gener-
ated from adult HPC sources, development of tissue protective ILC2 and NKp44+ ILC3 was notoriously
difficult.
Conclusions: Our data suggest that slow ILC recovery after allogeneic HCT may be related to an intrinsic inca-
pability of adult HPC to develop into ILC.
© 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Introduction lymphocytes that additionally have immune-regulating properties.

Graft-versus-host disease (GVHD) is a common complication of
allogeneic hematopoietic cell transplantation (HCT) that is associated
with high morbidity and mortality [1,2]. Tissue damage, induced, for
example, by conditioning chemotherapy and/or irradiation, plays an
important role in the development of mucositis and the pathophysi-
ology of GVHD. Tissue damage leads to activation and expansion of
donor alloreactive lymphocytes, resulting in a cascade of inflamma-
tion and more tissue destruction [3,4]. Tissue repair can be mediated
by innate lymphoid cells (ILCs), which are a family of tissue-resident
* Correspondence: Prof. dr. Mette D. Hazenberg, Department of Hematology,
Amsterdam UMC, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
E-mail address: m.d.hazenberg@amsterdamumc.nl (M.D. Hazenberg).
ILCs do not express antigen-specific receptors like T cells or B cells
and respond to environmental factors such as aryl hydrocarbon
receptor ligands, short chain fatty acids and cytokines [5,6]. In the
human intestine, which is one of the organs most affected in acute
GVHD, IL-22 producing ILC3 mediate tissue homeostasis and tissue
repair [7,8]. In addition, ILCs can have immunosuppressive properties
when expressing the ecto-enzymes CD39+ and CD73+. Ecto+ ILC3
metabolize adenosine triphosphate, released by damaged cells, into
adenosine that suppresses activation of co-cultured T cells [9]. Other
ILC subsets include interferon-g producing ILC1, and ILC2 that pro-
duce “type 2” cytokines like interleukin (IL)-5, IL-9, IL-13 and the tis-
sue protective factor amphiregulin [10 12].
The tissue healing and immune modulating capacities of ILCs have
led to studies investigating their role in the context of GVHD. In a

https://doi.org/10.1016/j.jcyt.2024.01.013
1465-3249/© 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/)
 S.Z. Omar et al. / Cytotherapy 26 (2024) 334 339
previous study, we observed that patients with relatively high fre-
quencies of circulating activated ILCs before and/or after allogeneic
HCT had a lower risk to develop mucositis and acute GVHD [13,14].
Moreover, patients who developed acute or chronic GVHD had lower
numbers of circulating ILCs, and biopsies of GVHD-affected tissues
demonstrated a significant reduction of ILC in these tissues [9].
Rapid reconstitution of ILC after chemotherapy-induced depletion
and after allogeneic HCT thus seems favorable. However, ILC recovery
after allogeneic HCT, in fact, was slow, and ILC numbers did not reach
normal levels until 6 months’ post-transplantation [13].
The requirements and mechanisms determining ILC reconstitu-
tion remain largely unknown. We hypothesized that, on the one
hand, expansion of mature ILCs present in the graft may contribute
to post-transplantation ILC reconstitution. By investigating the com-
position of adult HCT grafts, we showed that the presence of mature
ILCs in the graft was associated with ILC recovery after transplanta-
tion. Patients with greater-than-average proportions of ILCs in the
graft had a reduced risk to develop acute GVHD after transplantation
[14]. On the other hand, or in addition, ILC reconstitution may rely on
the development of ILCs from graft-derived multipotent hematopoi-
etic progenitor cells (HPCs). It is known that ILCs develop from com-
mon lymphoid progenitors in the bone marrow. Differentiation of
common lymphoid progenitors into mature ILC requires the tran-
scription factors DNA-binding protein inhibitor ID2, nuclear factor
interleukin-3 regulated protein, the cytokines IL-7 and stem cell fac-
tor (SCF) and cell surface expression of Notch ligands [15 17].
In the present study, we investigated the potential of human HPCs
obtained from fetal, neonatal and adult sources to develop into
mature ILC subsets. We established an ex vivo differentiation model
allowing for simultaneous differentiation of HPCs into all helper ILC
subsets. We found that independent of the source, HPCs were able
to give rise to all helper ILC subsets ex vivo, although the relative
frequencies of ILC that developed in this model differed between HPC
sources.
Methods
Human cells
Study protocols were approved by the Medical Ethical Committee
of the Amsterdam University Medical Centers location AMC and San-
quin Blood Supply (Amsterdam, The Netherlands). Human fetal liver
was obtained from elective abortions (14 20 weeks of gestational
age) and umbilical cord blood from term deliveries. Adult bone mar-
row cells were obtained from patients who underwent sternotomy
for thoracic surgery. Aliquots of granulocyte colony-stimulating fac-
tor mobilized peripheral blood allogeneic and autologous HCT grafts
were collected from apheresis materials [14]. Fetal liver tissues were
mechanically disrupted using the Stomacher 80 Biomaster (Seward,
West Sussex, UK). Mononuclear cells were isolated by Ficoll-Paque
density gradient centrifugation (Lymphoprep; GE Healthcare, Chi-
cago, IL, USA), CD34+ cells purified (CD34 MicroBead kit; Miltenyi
Biotec, San Diego, CA, USA), frozen in 20% FCS containing 10% DMSO
and kept in liquid nitrogen.
HPC culture
OP9 murine stromal cells transfected with Notch ligand Jagged 1
(OP9-JAG1) were maintained in Minimum Essential Medium a
medium (Invitrogen, Waltham, MA, USA) supplemented with 8% fetal
calf serum and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis,
MO, USA). Aliquots of CD34-positive hematopoietic stem cells were
thawed, stained with fluorochrome-conjugated antibodies (supple-
mentary Table 1) and LIVE/DEAD Fixable Green Dead Cell Stain (Invi-
trogen), and sorted with a FACSAria (Becton Dickinson). Flow
cytometry purified human live, lineage (lin) CD3 CD34+CD45RA+
335
and lin CD3 CD34+CD45RA HPCs were sorted, and 5.000 HPCs
were seeded on to 5.000 OP9-JAG1 cells in Minimum Essential
Medium a supplemented with 20% fetal calf serum, p/s, 10 Units/mL
recombinant human (rh)IL-2 (BioLegend, San Diego, CA, USA),
10 ng/mL rhIL-7 (Tebubio, Le Perray-en-Yvelines, France), 10 ng/mL
rhSCF (Invitrogen) and 10 ng/mL rhFlt3L (Tebubio; Figure 1A).
Cells were transferred to freshly plated OP9-JAG1 cells once a week.
Cytokines were replenished twice a week.
Flow cytometry and cytokines
Intracellular cytokine production was measured by stimulating
the cells with 10 ng/mL phorbol myristate acetate (Sigma) and
500 nmol/L Ionomycin (Merck, Rahway, NJ, USA) for 6 hours at 37°C,
with GolgiPlug (BD Biosciences, San Diego, CA, USA) added for the
final 4 hours. To measure intracellular transcription factors or cyto-
kines, cells were incubated with Zombie UV Dye (BioLegend) and cell
surface antigen monoclonal antibodies, incubated with Fixation
Buffer and Intracellular staining Perm Wash Buffer (Thermo Fisher
Scientific, Waltham, MA, USA) and stained with transcription factor/
cytokine antibodies. Data were acquired with a LSRII flow cytometer
(Becton Dickinson) and analyzed using FlowJo software.
Quantitative real-time polymerase chain reaction (PCR)
Quantitative PCR was performed on RNA of CD45+Lin CD127+
cells that were sorted after 4 weeks of culture. RNA was isolated
using the NucleoSpin RNA XS kit (Macherey-Nagel, Du € ren, Nordr-
hein-Westfalen, Germany) and reversed transcribed into comple-
mentary DNA (cDNA) using the High Capacity cDNA Archive Kit
(Applied Biosystems, Waltham, MA, USA). Quantitative PCR was per-
formed to measure expression of the Tbx21, Gata3 and Rorc genes in
a CFX Connect Real-Time System using IQ SYBR Green Supermix
(both Bio-Rad, Hercules, CA, USA). Primer sets are listed in supple-
mentary Table 2. Expression was analyzed using Bio-Rad CFX Man-
ager 3.1 software and normalized to the expression of
glyceraldehyde 3-phosphate dehydrogenase.
Statistical analysis
Results are presented as median plus interquartile range. To cal-
culate statistical significance, the Wilcoxon signed-rank test or Man-
n Whitney U test was used (GraphPad Prism software). *P < 0.05
and **P < 0.01 were considered statistically significant.
Results
ILC can be generated from CD34+ HPCs
First, we set up a protocol to differentiate CD34+ HPCs into mature
ILC ex vivo. We sorted CD34+ HPC populations from healthy human
fetal liver by fluorescence-activated cell sorting based on the differ-
ential expression of CD45RA into common CD34+CD45RA HPCs and
early lymphoid progenitor CD34+CD45RA+ HPCs (Figure 1A) [18,19].
Each HPC subset was co-cultured with OP9 mouse stromal cells that
constitutively expressed the Notch ligand Jagged-1 (OP9-JAG1), as
Notch signaling has been shown to contribute to ILC development
[20]. Cultures were supplemented with a differentiation and expan-
sion cytokine cocktail that included IL-7, SCF, FLT3L and/or IL-2, up to
28 days (Figure 1B). Flow cytometric analysis was performed on days
14, 21 and 28. Mature ILCs were defined as live, lineage negative
(lin ) CD45+CD3 CD127+ lymphocytes. Using this protocol, both HPC
subsets were able to differentiate into ILC, but the percentage of ILCs
obtained from CD34+CD45RA+ HPC was greater than that derived
from CD34+CD45RA HPC (Figure 1C). For follow-up experiments, we
therefore focused on CD34+CD45RA+HPC.
336 S.Z. Omar et al. / Cytotherapy 26 (2024) 334 339

Figure 1. Generation of ILC and ILC subsets from CD34+ HPCs. (A) Representative flow cytometry dot plot of CD34+CD45RA and CD34+CD45RA+ HPC derived from human fetal liver.
The lineage (lin) antibody cocktail included antibodies directed against CD1a, CD4, CD5, CD14, CD16, CD19, CD94, CD123, BDCA2, TCRab, TCRgd and FcƐR1a. (B) Experimental setup
to culture purified CD34+CD45RA and CD34+CD45RA+ HPC on OP9-JAG1 expressing stromal cells for 28 days in the presence of differentiation and expansion recombinant human
cytokine cocktails as indicated. (C) ILC development from CD34+CD45RA or CD34+CD45RA+ HPC after 14 (n = 5), 21 (n = 8) and 28 (n = 8) days of culture. (D) ILC subsets derived
from fetal liver HPC (n = 8) at day 28. ILC subsets are defined as CRTH2 CD117 NKp44 ILC1, CRTH2+ ILC2 and CRTH2 CD117+ NKp44 or NKp44+ ILC3. (E) Frequency of transcrip-
tion factors analyzed by flow cytometry (n = 5) or (F) qPCR (n = 5). (G) Flow cytometry analysis of intracellular cytokine expression (n = 4). Data represent mean plus standard devia-
tion of n independent experiments. *P < 0.05; **P < 0.01 by Wilcoxon signed-rank test. (Color version of figure is available online.)

To evaluate which helper ILC subsets were generated ex vivo,
we analyzed the phenotype of ILC. Lin CD45+CD127+ helper
ILC subsets were defined by distinct cell-surface markers as
CRTH2 CD117 NKp44 ILC1, CRTH2+ ILC2 and NKp44 or NKp44+
CRTH2 CD117+ ILC3. All ILC subsets differentiated from
CD34+CD45RA+ fetal liver HPC in culture, with the lowest frequency
being observed for ILC1 (Figure 1D). To confirm that cells generated
were bona fide ILC, we evaluated signature transcription factor
expression by flow cytometry (Figure 1E and supplementary Figure
1A) and quantitative PCR (Figure 1F). ILC1s are defined by the expres-
sion of Tbet, ILC2 express GATA3 and ILC3 are characterized by the
expression of RORg t. We found that most of the CD127+ ILC
expressed RORg t, consistent with the fact that most cells expressed
an ILC3 phenotype (supplementary Figure 1B and Figure 1E). Expres-
sion of GATA3 was detected in 25% of the ILC in line with the percent-
age of ILC2 generated in the cultures. Expression of T-bet, which is
associated with ILC1, and EOMES, associated with natural killer cells,
was lower compared with RORg t and GATA3 (Figure 1E), which is
also consistent with the ILC phenotype distribution (Figure 1D).
Finally, to evaluate the functional capacity of ex vivo generated
ILC subsets, we analyzed signature cytokine production in
lin CD127+EOMES helper ILC after restimulation with phorbol
 S.Z. Omar et al. / Cytotherapy 26 (2024) 334 339 337

myristate acetate and ionomycin (Figure 1G). We observed intracel-
lular expression of interferon-g , IL-13, IL-17 and IL-22, which is asso-
ciated with the presence of ILC1, ILC2 and ILC3, respectively
(Figure 1G). Taken together, these data demonstrate that HPCs iso-
lated from human fetal liver are able to differentiate into all mature
helper ILC subsets ex vivo and express signature transcription factors
and effector cytokines.
Generation of ILCs from HPC differs between sources
Having established an ex vivo model for development of all ILC
subsets, we next investigated whether HPCs derived from sources
that are used for allogeneic HCT are also able to differentiate into
mature ILC subsets in this model. Bone marrow aspirates, umbilical
cord blood samples, peripheral blood granulocyte colony-stimulating
factor mobilized autologous and allogeneic HCT grafts all contained
CD34+CD45RA and CD34+CD45RA+ HPC, albeit at different ratios
(Figure 2A). In all sources, the frequency of CD34+CD45RA HPC was
greater than the frequency of CD34+CD45RA+ HPC. We sorted
CD34+CD45RA+ HPC from each source and cultured the cells for
28 days ex vivo as described for fetal liver derived HPCs (Figure 1B).
We observed that HPCs of all sources had the capacity to differentiate
into mature ILCs (Figure 2B). However, significantly greater propor-
tions of ILCs were generated from fetal liver and umbilical cord blood

Figure 2. Generation of ILCs from HPC differs between stem cell sources. (A) Frequency of CD34+CD45RA and CD34+CD45RA+ HPCs present in adult G-CSF mobilized peripheral
blood allogeneic HCT grafts (ALLO) (n = 4), autologous HCT grafts (AUTO) (n = 4), adult bone marrow (BM) (n = 3), umbilical cord blood (UCB) (n = 3) and fetal liver (FL) (n = 7). (B)
Frequency of ILC and (C) frequency of ILC subsets obtained from fetal, neonatal and adult (n = 4) CD34+CD45RA+ HPC sources after 28 days of culture. Data represent mean and stan-
dard deviation of n independent experiments. *P < 0.05; **P < 0.01 by non-parametric paired Wilcoxon t-test (A) or Mann Whitney U test (B-C). G-CSF, granulocyte colony stimu-
lating factor. (Color version of figure is available online.)
338 S.Z. Omar et al. / Cytotherapy 26 (2024) 334 339
as compared with adult bone marrow and mobilized autologous
or allogeneic HCT grafts. A similar trend was observed when we
cultured CD34+CD45RA HPCs (supplementary Figure 2). All helper
ILC subsets were generated independent of the source of HPC, but
the relative frequencies of ex vivo generated ILC subsets did
differ depending on the source of HPC (Figure 2C and supplementary
Figure 2B). Umbilical cord blood derived HPC differentiated mostly
into NKp44+ ILC3 and NKp44 ILC3, whereas adult bone mar-
row derived HPC mostly gave rise to ILC1 and NKp44 ILC3. HPCs
from autologous HCT grafts predominantly differentiated into
NKp44 ILC3. Unfortunately, the number of ILCs that differentiated
from HPCs sorted from allogeneic HCT grafts was too low to allow for
reliable ILC subset analysis. We conclude that ILCs can be generated
ex vivo from all HPC sources but that the relative frequencies of ILC
subsets generated from these HPC do depend on the source of HPCs.
Discussion
Donor-derived ILCs likely play an important role in protecting tis-
sues of allogeneic HCT recipients against the development of tissue
damage and acute GVHD [9,13,14,21 23]. Similar to T cells, however,
ILCs have slow reconstitution dynamics after allogeneic HCT
[13,14,21,24 26]. T-cell reconstitution after transplantation is a
biphasic process that initially, in the first few months after transplan-
tation, is dominated by expansion of mature T cells that were present
in the graft. Later, naive T cells that are produced de novo from trans-
planted donor HPC in the thymus enter the circulation and contribute
to a more diverse T-cell population [27 29]. It can be envisioned that
ILC reconstitution follows a similar dynamic. We previously demon-
strated that mature ILCs are present in allogeneic HCT grafts and that
the proportion of ILC in the grafts tended to correlate with ILC recon-
stitution after transplantation [14]. To what extend de novo develop-
ment of ILC from HPC contributes to ILC reconstitution in allografted
recipients remains to be determined.
Here, we investigated the capacity of human HPCs derived from
sources used for transplantation, such as mobilized peripheral blood,
bone marrow and umbilical cord blood, to differentiate into mature
ILCs. For this, we set up an ex vivo differentiation model using fetal
liver HPC that allows for simultaneous development of all helper ILC
subsets, using a Notch ligand expressing mouse stromal cell line.
Both CD34+CD45RA and CD34+CD45RA+ fetal liver HPCs were able
to differentiate into ILC, although CD34+CD45RA+ HPC were more
potent, which may be related to the fact that CD34+CD45RA+ HPC are
more committed to the lymphoid lineage than CD34+CD45RA HPC
[18,19]. We found that under these culture conditions HPC isolated
from pre- and neonatal sources, i.e., fetal liver and umbilical cord
blood, have a better capacity to differentiate toward ILCs than HPC
from adult sources, which yielded significantly fewer ILCs. This is in
line with previous findings, where HPCs were cultured under
stroma-free conditions [30]. Together, these data suggest that if ILC
development is dependent on the age of the HPCs, and we assume
that de novo generation of ILC from transplanted HPCs is an important
contributor to ILC reconstitution after allogeneic HCT, ILC recovery
may be better after transplantation with CB-derived HPCs compared
with transplantation of adult donor derived HPCs. In a small study
performed among a diverse cohort of pediatric and adult allogeneic
HCT recipients, however, no clear differences in ILC reconstitution
were observed between pediatric patients who received cord blood
or adult HPCs [21]. The observation in this study that ILC2 reconstitu-
tion was significantly slower in adults receiving adult HPCs compared
with pediatric allogeneic HCT recipients who received cord blood
HPC [21] does suggest that age-related factors play a role in ILC2
reconstitution. In mice, ILC2 were reported to harbor abortive T-cell
receptor (TCR)g rearrangements, but no other TCR transcripts, and
intracellular CD3 [31,32]. In human fetal thymus, CD5+ ILC progeni-
tors have been identified [33]. Although genomic TCR loci in human
ILC subsets have not been evaluated to date, these data raise the
hypothesis that human ILC development may also rely on the thy-
mus, similar as naive T-cell development. Since thymic activity at
adult age is notorious low, this may provide an additional explana-
tion for the slow recovery of ILC2 after allogeneic HCT.
ILC3s were generated from HPCs at greater frequencies than ILC2,
irrespective of the HPC source. This is in line with the observation
that in adults who received allogeneic HCT with adult HPC ILC3
reconstitution was relatively fast, when compared with ILC2, with
relatively high numbers of circulating ILC3 compared with numbers
obtained from healthy individuals [13]. Altogether, these findings
support the hypothesis that early ILC reconstitution after allogeneic
HCT with adult HPC may depend more on expansion of mature ILCs
present in the graft rather than de novo generation of ILCs from HPCs,
in analogy with post-transplantation T-cell reconstitution.
In conclusion, stem cell grafts contain HPCs that are able to differenti-
ate into mature ILCs. These results are important because better under-
standing of ILC reconstitution may pave the way for novel approaches to
improve ILC reconstitution, such as adoptive transfer of mature ILC,
which may help to prevent GVHD in allogeneic HCT recipients.
Declaration of Competing Interest
The authors have no commercial, proprietary or financial interest
in the products or companies described in this article.
Funding
This study was supported by a VIDI grant (NWO ZonMW
#91715362; M.D.H.) and a LSBR Fellowship (1438F; M.D.H.).
Author Contributions
Conception and design of the study: BB and MDH. Acquisition of
data: SZO, NJEH and VvH. Analysis and interpretation of data: SZO,
NJEH and VvH. Drafting or revising the manuscript: SZO, NJEH, VvH,
JMRvdM, CV, BB and MDH. All authors have approved the final
article.
Acknowledgments
The authors thank Esther Siteur van Rijnstra and Cynthia van der
Linden of the Mice Facility for providing the OP9 stromal cells, and
fetal liver CD34+ HPC. The authors also thank the Flow Cytometry
Facility of the Amsterdam UMC, location AMC, department of EXIM
for their excellent technical support.
Data Availability
Data and protocols will be made available upon written request to
the corresponding author.
Supplementary materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.jcyt.2024.01.013.
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