Abstract
Retinal progenitor cells (RPCs) are the source of all retinal cell
types during retinogenesis. Until now, the isolation and expansion of
RPCs has been at the expense of their multipotency. Here, we report
simple methods and media for the generation, expansion, and
cryopreservation of human induced pluripotent stem-cell derived-RPCs
(hiRPCs). Thawed and passed hiRPCs maintained biochemical and
transcriptional RPC phenotypes and their ability to differentiate into
all retinal cell types. Specific conditions allowed the generation of
large cultures of photoreceptor precursors enriched up to 90% within a
few weeks and without a purification step. Combined RNA-seq analysis
between hiRPCs and retinal organoids identified genes involved in
developmental or degenerative retinal diseases. Thus, hiRPC lines could
provide a valuable source of retinal cells for cell-based therapies or
drug discovery and could be an advanced cellular tool to better
understand retinal dystrophies.
Subject terms: Multipotent stem cells, Stem-cell differentiation
__________________________________________________________________
An in vitro method can expand and bank retinal progenitor cells from
hiPSC-derived retinal organoids, representing a potential source of
retinal cells for future cell-based therapies or drug discovery models.
Introduction
In mammals, neuroregenerative capacity is poor, including that of the
human retina. Therefore, retinal dystrophies (RDs) that cause the
definitive loss of neural cells typically result in permanent visual
impairment. Preventing degeneration and rescuing the degenerated retina
are major challenges for which stem cell-based therapies show
promise^[44]1,[45]2. Many methods have emerged to generate human
induced pluripotent stem cell (hiPSC)-derived retinal cells and
tissues, such as retinal organoids (ROs), for cell therapy, drug
discovery^[46]3,[47]4, or to better understand inherited RDs by
modeling the progression of degeneration. However, the selection of
specific cells within ROs requires sorting, limiting the scale-up of
production. Part of the challenge of using hiPSC derivatives in
cell-based therapies is the production of a large number of specific
and identical cells. Because new therapies are cell-intensive, the
simple and efficient production of such cells is required. To meet
these needs, we proposed the use of mitotic and multipotent retinal
progenitor cells (RPCs) isolated from early ROs. Indeed, RPCs are the
source for the generation of all retinal cell types during
retinogenesis^[48]5. Initially isolated from human fetal retinas^[49]6,
RPCs can be generated from differentiation protocols using human
iPSCs^[50]7–[51]11. Although RPCs appear to be expandable, numerous
studies have described the loss of their multipotency during
culture^[52]6 or following multiple passages^[53]12–[54]14. Here, we
describe a method and an innovative RPC-dedicated medium (RPCM) that
allow the amplification of hiPSC-derived RPCs (hiRPCs) in adherent
culture condition while maintaining their multipotency. The RPCM-based
culture contains five extrinsic factors (CHIR99021, PURMORPHAMINE, ATP,
FGF2, and EGF) that act on pathways involved in retinal development and
allowed several passages of hiRPCs for the production of millions of
cells from cryopreserved stocks. RNAseq analysis confirmed preserved
multipotency, with a preneurogenic phenotype, of hiRPCs expanded in
RPCM. From cryopreserved stocks, hiRPCs were able to differentiate into
all retinal cell types, such as retinal pigmented epithelium (RPE),
retinal ganglion cells (RGCs), horizontal cells, amacrine cells and
photoreceptor precursor cells (PPCs) within a few weeks as well as
mature photoreceptors expressing rod and cone opsins, bipolar cells and
Müller glial cells in long-term culture. Moreover, we describe defined
culture conditions that allow the generation of enriched cultures of
hiRPC-derived PPCs (hiPPCs) of up to 90% in 2 weeks without a
purification step. Thus, we describe methods and media to isolate,
expand, and cryopreserve bankable hiRPCs as a powerful cellular tool.
This multipotent cell line, as well as cell derivatives, could be used
for developmental research, cell-based therapy, and drug discovery in
the foreseeable future.
Results
Generation of the human iPSC-derived retinal progenitor cell line
ROs were generated from the human fluorescent reporter
AAVS1::CrxP_H2BmCherry-hiPSC-5FC line^[55]15 and from the
nonfluorescent hiPSC-2^[56]16 and hiPSC-5F^[57]17 lines using our
previously reported protocol^[58]16,[59]18 (see materials and methods).
Data presented in main figures were done with the fluorescent reporter
hiPSC-5FC line to target hiPPCs through the mCherry expression. Thus,
after 4 weeks (W4) of hiPSC differentiation, ROs were picked and
cultured under floating-culture conditions until W6 (Fig. [60]1a).
Human iPSC-derived RPCs were isolated from ROs at W6, which are
composed of two major cell types organized in two layers (Fig. [61]1b).
The internal layer contains PAX6^+/VSX2^- cells, mainly corresponding
to RGCs, the first post-mitotic differentiated cell type^[62]7,[63]16
(Supplementary Fig. [64]1). The external layer is composed of a cell
population co-expressing PAX6^+/VSX2^+ and the mitotic marker KI67,
representing RPCs (Fig. [65]1b, Supplementary Fig. [66]2). RT-qPCR
analysis confirmed that the progenitors present in ROs at W6 still
expressed the eye-field transcription factors (EFTFs) RX, PAX6, SIX6,
SOX2 and the optic vesical specific transcription factor VSX2 while
retaining the expression of the photoreceptor marker CRX at a basal
level relative to ROs at W7 (Fig. [67]1c). Moreover, the absence of
endogenous mCherry staining, illustrating the absence of cells
committed towards the photoreceptor lineage, confirmed the
undifferentiated state of the RPC layer at W6 (Fig. [68]1b,
Supplementary Fig. [69]2). Before RPC isolation, the pigmented portion
of W6 ROs was removed to avoid any RPE contamination in the downstream
hiRPC cultures (Fig. [70]1a). This cleaning step is not possible before
W6 because of the small size of the ROs and the absence of the
pigmentated portion. After papain-based dissociation of cleaned ROs,
seeded cells were designated as hiRPCp0 and cultured in a new specific
RPC-dedicated medium (RPCM) that acts on multipotency and proliferation
by the addition of the five extrinsic factors CHIR99021, PURMORPHAMINE,
FGF2, EGF, and ATP in a growth factor-free basal medium (BM) used for
pluripotent stem-cell cultures^[71]19 (Fig. [72]1a). Moreover, only the
RPCM containing all molecules was able to maintain simultaneously the
proliferation and the expression of the key RPC transcription factors
VSX2, PAX6 and RAX (Supplementary Fig. [73]3). After 1 week in culture
(W1), homogeneous and adherent hiRPCp0 still expressed VSX2, PAX6, and
KI67 but not mCherry, as observed in W6 ROs (Fig. [74]1b and d,
Supplementary Fig. [75]3). At W1, hiRPCp0 contained 50,2 ± 6,6% of
KI67^+ cells. RT-qPCR analysis confirmed EFTF expression and the
positive selection of hiRPCs by the low expression of CRX
(Fig. [76]1e). Moreover, pluripotency markers were shutdown in hiRPCs
comparatively to hiPSCs (Supplementary Fig. [77]4). To bank retinal
progenitors, hiRPCp0 were passed and expanded for 1 week in RPCM and
hiRPCp1 were cryopreserved in stem cell-dedicated cryopreservation
medium at 5 × 10^6 cells/ml (Fig. [78]1a). Thus, this culture condition
positively selected the hiRPCs which are the only mitotic cells at the
expense of the the first differentiated post-mitotic cells. We
investigated the possibility of passing thawed hiRPCs once a week to
increase hiRPC production (Fig. [79]1f). One million thawed cells
cultured in RPCM produced 4.6 ×10^6 ± 0.6 hiRPCp2 in W1, 14.5 ×10^6
hiRPCp3 in W2, and 43.23 ×10^6 ± 2.96 in W3 (Fig. [80]1g). The mean
multiplication factor between hiRPCp2 and hiRPCp4, was 3.6 ± 0.9
(Fig. [81]1g). Under RPCM culture conditions, the expression of EFTFs
by W1-expanded hiRPCs was stable after passaging (Fig. [82]1h) and
immunostaining confirmed the homogenous co-expression of VSX2, PAX6,
KI67, and RAX in all cells (Fig. [83]1i, Supplementary Fig. [84]5). In
addition, as expected, mCherry staining, which identifies CRX
expression, was at background levels (Fig. [85]1i, Supplementary
Fig. [86]5). Depending on the hiPSC line used to generate hiRPCs, EFTF
expressions can decrease at passage 4 as observed in hiPSC-2-derived
hiRPCs but without affecting their differentiation potential
(Supplementary Fig. [87]6). After thawing, hiRPCs cultured in RPCM grew
as bright cell clusters (Fig. [88]1i) and this morphology was lost for
the neural phenotype when the cells are cultured in BM (Supplementary
Fig. [89]7). Proliferation of hiRPCs was up to 10-fold higher in RPCM
that in BM (Supplementary Fig. [90]7). Evidence for multipotency under
RPCM culture conditions was shown by RT-qPCR, with enhanced expression
of RAX, PAX6, VSX2, SOX2, and SIX6 and the maintenance of CCND1
expression, which sustains proliferation (Supplementary Fig. [91]7). On
the contrary, in BM, EFTF and CCND1 expression decreased, allowing
hiRPC differentiation, highlighted by the upregulation of photoreceptor
(CRX) and retinal ganglion cell (BRN3A) markers (Supplementary
Fig. [92]7).
Fig. 1. Generation and characterization of a retinal progenitor cell line
from iPSC-derived retinal organoids.
[93]Fig. 1
[94]Open in a new tab
a Schematic diagram illustrating the protocol for the generation of
RPCs from hiPSCs and cell banking. b Immunofluorescence staining of
cryosectioned ROs at W6 for VSX2, PAX6, Ki67, and endogenous mCherry
staining. c RT-qPCR analysis of eye-field transcription factors (EFTFs;
RAX, PAX6, VSX2, SIX6), SOX2, and CRX in ROs between W4 and W7. Data
are normalized to that of W4 ROs and presented as mean ± SD (n = 3 per
time point). d Immunofluorescence staining of hiRPCp0 after one week of
culture (W1) in RPCM for VSX2, PAX6, and Ki67 and endogenous mCherry
staining. e RT-qPCR analysis of EFTFs (RAX, PAX6, SOX2, SIX6), VSX2,
and CRX in ROs at W4 and W6, dissociated ROs at day 1, and hiRPCp0
expanded for one week (W1) in RPCM. Data are normalized to that of W4
ROs and presented as mean ± SD (n = 3 per time point). f Schematic
diagram illustrating hiRPC expansion in RPCM from cryopreserved hRPCp1
to hiRPCp4 in three weeks. g Multiplication factor (red) and hiRPC
number (gray histograms) after successive passages. Data are normalized
to that of seeded hiRPCp2 at D0 and presented as mean ± SD (n = 6 for
hiRPCp2 and n = 3 for hiRPCp3-4). h RT-qPCR analysis of EFTFs (RAX,
PAX6, SIX6, SIX3, LHX2) and VSX2 in W4 ROs and hiRPCp2 to hiRPCp4. Data
are normalized to that of W4 ROs and presented as mean ± SD (n = 3 per
time point). i hiRPC characterization after one week (W1) of culture in
RPCM by phase-contrast and brightfield microscopy (left panels) and
immunofluorescence staining of hiRPCp2, hiRPCp3, and hiRPCp4 for VSX2,
PAX6, and Ki67 and endogenous expression of mCherry. One-way ANOVA
followed by a Dunnett’s multiple comparison test (c, e, g, h).
Comparison to W4 ROs (c, e, h) or hiRPCp2 (g). ***p < 0.001;
**p < 0.01; *p < 0.05. Nuclei were counterstained with DAPI (blue).
hiPSC-5FC-derived cells. Scale bar: b, d, i, 50 µm.
Whole transcriptome analysis of hiRPCs
We characterized the multipotency of the hiRPC line by comparing whole
transcriptome sequencing (RNAseq analysis) of fresh hiRPCp0, thawed
hiRPCp2, and passed hiRPCp3 and hiRPCp4 to W4 and W6 ROs. W4 ROs were
mainly composed of RPCs and W6 ROs of both RPCs and RGCs (Supplementary
Figs. [95]1 and [96]2). In total, 16,168 transcripts were detected
(Supplementary Data [97]3) and those with a TPM expression value ≥ 10
were considered to be clearly expressed. This threshold was determined
based on CRX expression in W4 ROs, when CRX was present at a very low
level (2.8 TPM), whereas, in W6 ROs, CRX expression was higher,
reflecting the progression of retinal differentiation (10.2 TPM)
(Fig. [98]1c, Supplementary Data [99]3). Principal component analysis
(PCA) showed four clusters based on the first principal component
(PC1), illustrating a different transcriptional landscape between W4
ROs, W6 ROs, hiRPCp0, and other hiRPC passages (Fig. [100]2a). PC2
analysis supported the phenotypic reversion of the RPC population,
composed of hiRPC clusters, to a transcriptome very close to that of
early W4 ROs (Fig. [101]2a, bleu arrow). Heatmap analysis of EFTF
expression confirmed high and stable expression of early retinal
differentiation factors, such as PAX6, RAX, VSX2, SIX3, SIX6, and LHX2,
in each sample (Fig. [102]2 b, top panel), as previously shown by
RT-qPCR (Fig. [103]1h). We then focused on the expression of genes
described to be involved in the multipotency or neurogenic property of
retinal progenitors^[104]20–[105]22 (Fig. [106]2b). As expected, the
expression of multipotency markers tended to decrease in ROs between W4
and W6, whereas that of neurogenic genes increased, reflecting their
early commitment to differentiation (Fig. [107]2b, middle panel).
Nevertheless, the expression of these genes in RPCM-expanded hiRPCs was
generally similar to the expression level observed in W4 ROs
(Fig. [108]2b). This result is consistent with those of the PCA,
confirming that the expanded RPC population corresponded to retinal
progenitors at a similar progenitor stage as that found in W4 ROs.
Remarkably, the expression of specific multipotency genes, such as
GJA1, PFN1, IGFBP5, ATP1A2, and FGF19, increased under RPCM culture
conditions, supporting the stemness characteristic of hiRPCs
(Fig. [109]2b). The differential expression of selected multipotency
genes (GJA1, IGFBP5, ATP1A2, CCND2, BMP7) and neurogenic genes (BASP1,
ATHO7, MAP1B, SPP1, FOXN4, GADD45A) was confirmed by qRT-PCR
(Supplementary Fig. [110]8). To better decipher the differences between
RPCs in early ROs and expanded hiRPCs, we first compared the
transcriptome of W4 ROs to that of hiRPCp2 to identify genes expressed
by both groups or specifically by only one (Fig. [111]2c). These two
groups showed 84.4% (9,619, Supplementary Data [112]4a) of their
expressed genes to be in common. Based on our filtering criteria, 10.1%
(1,153, Supplementary Data [113]4b) and 5.5% (622, Supplementary
Data [114]4c) were considered to be specifically expressed in W4 ROs
and hiRPCp2, respectively (Fig. [115]2c). Filtering of the
differentially expressed genes (DEGs) between hiRPCp2 and W4 ROs with a
fold change (FC) ≥ 2 or ≤ 2, a false discovery rate (FDR) ≤ 0.05, and
minimum expression of 10 TPM led to the identification of 940 genes
(Supplementary Data [116]4d). Pathway enrichment analysis of these DEGs
and circular representation clearly showed the deregulation of pathways
related to cell-cycle activity (GO:0006260; GO:0044772; GO:0000793;
R − HSA − 1640170) and brain and eye development (GO:0007420;
GO:0001654), with mostly upregulated genes, based on the calculated
z-score. By contrast, the progression of W4 ROs toward hiRPCp2 occurred
without promoting differentiation (GO:0070848; GO:0045664; GO:0007423;
GO:0007423; GO:0048598; GO:0010001; R − HSA − 9675108), with the
expression of most of the genes downregulated (Fig. [117]2d,
Supplementary Data [118]4e). This analysis supports reinforcement of
the multipotent and proliferative phenotype of hiRPCs cultivated in
RPCM. We then filtered the results based on a FC ≥ 2 before Metascape
analysis to focus the GO term enrichment on upregulated genes only in
W4 ROs and hiRPCp2 (Fig. [119]2d). Thus, 325 genes were selected for
hiRPCp2 (Fig. [120]2e, Supplementary Data [121]4f) and 788 for W4 ROs
(Fig. [122]2f, Supplementary Data [123]4g), showing mainly variations
in structural components of the cells that this could be a result of
changing from free floating to adherent culture conditions.
Fig. 2. Transcriptomic characterization of hiRPCs by RNAseq analysis.
[124]Fig. 2
[125]Open in a new tab
a Principal component analysis (PCA) of W4 ROs, W6 ROs, hiRPCp0, and
hiRPCp2–4. Each point represents one sample and biological replicates
are shown in the same color. b Hierarchical clustering of EFTFs and
multipotent and neurogenic gene markers in W4 ROS, W6 ROS, hiRPCp0, and
hiRPCp2–4. hiRPCs were expanded for one week in RPCM. c Venn diagram of
genes expressed in W4 ROs and hiRPCp2 with TPM ≥ 10. d Circular
visualization (left panel) and table of the over-represented GO
pathways of interest (right panel) identified with Metascape using the
DEGs between hiRPCp2 and W4 ROs with a FC ≥ 2, FDR ≤ 0.05, and TPM ≥ 10
in both groups. Red dots (upregulated genes) and blue dots
(downregulated genes) represent an overview of regulated genes in
hiRPCp2 relative to W4 ROs. Z-score bars indicate whether an entire
biological process is more likely to be increased or decreased based on
the genes within it. e Gene ontology (GO) enrichment analysis for
upregulated genes with fold change (FC) ≥ 2 in hiRPCp2. f GO enrichment
analysis for upregulated genes with FC ≥ 2 and FDR ≤ 0.05 in W4 ROs. g
Heatmap of genes with R = (W4 RO TPM) / (W6 RO TPM) > 5. Black dots
indicate genes associated with microphthalmia or retinal dystrophies
(RDs). hiPSC-5FC-derived cells.
hiRPCs as a tool for retinogenesis studies
The maintenance of multipotency of the hiRPC population from W6 ROs
during selection expansion in RPCM provides an innovative tool to
trigger the expression of genes involved in the early stages of
retinogenesis. We selected genes for which the expression differs (FC ≥
2) in ROs between W4 and W6 and returns to their initial levels (as in
W4 ROs) in expanded hiRPCp2 (Supplementary Fig. [126]8). Such an
approach led to the identification of 617 restored genes, potentially
representing actors involved in the maintenance of multipotency, as
well as early neurogenesis (Supplementary Data [127]5a). To select
genes potentially involved in the multipotency of RPCs, we ranked them
by their expression ratio (R), representing fold-expression changes of
genes between W4 and W6 ROs (Supplementary Data [128]5b). R > 1 and
R < 1 identified genes for which the expression decreased or increased
between W4 and W6 ROs, respectively. A heatmap representing genes with
a ratio > 5 (Fig. [129]2g) confirmed the presence of genes already
known to be multipotency makers, such as GJA1, BMP7, IGFBP5, and CCND2,
validated by RT-qPCR (Supplementary Fig. [130]8). From these data, we
propose a short list of genes (VAX1, ALDH1A3, CLDN1, DSP, RAB38, VAMP5,
SMOC1, RAB29, ADAMTS9, ETNPPL, HAS2, ELK3, TAGLN, PRELP, CA2, KCNJ13,
LAMB1, GAS2L1, ABCA4, MAMDC2, and CD74) and one long ncRNA (lncRNA,
[131]AL035425.3) that could be considered as potential new markers of
RPC multipotency (Fig. [132]2g, Supplementary Data [133]5b).
Interestingly, genes involved in microphthalmia (VAX1, ALDH1A3, GJA1,
and SMOC1) and retinal dystrophies (ADAMTS9, KCNJ13, CA2 and ABCA4)
were also selected (Fig. [134]2g). Reciprocally, the R < 1 transcript
list contained genes involved in neurogenesis, such as MAP1B, SPP1,
FOXN4, and GADD45A, validated by RT-qPCR (Supplementary Fig. [135]8),
and in the early differentiation of RPCs, such as CRX, NEUROD1, and
POU4F1 (Supplementary Data [136]5b).
Neuroretinal cell production from hiRPCs
The next step was to demonstrate that our newly generated hiRPCs are
able to differentiate into the various retinal cell types. Thus,
proneural medium (ProNM) was used, as described for RO maturation in
floating culture^[137]18, to trigger retinal differentiation
(Fig. [138]1a). Although the differentiation of hiRPCs can be performed
at different passages (Supplementary Figs. [139]9 and [140]10), we used
here hiRPCp3 generated from thawed and expanded hiRPCp1 (Fig. [141]3a).
After 5 weeks (W5) in culture, hiRPCs differentiated mainly into
hiPPCs, identified by endogenous mCherry staining (71.4% ± 8.4), with
among hiPPCs rod photoreceptor precursors co-expressing mCherry and NRL
(46.7% ± 4.8) (Fig. [142]3b, c). Nonphotoreceptor cells expressing PAX6
represented 26.6% ± 6.7, corresponding to amacrine cells (PAX6^+/AP2^+,
25.7% ± 6.8) and horizontal cells (PAX6^+/LHX1^+, 2.9 ± 1.32)
(Fig. [143]3b, c). RT-qPCR confirmed increased expression of these
specific cell markers (CRX, NRL, AP2, and LHX1) during culture from W1
to W5 (Fig. [144]3d). To accelerate and promote hiPPCs generation, we
cultured hiRPCs for 3 weeks in ProNM containing the NOTCH inhibitor
DAPT, previously reported to promote photoreceptor differentiation
within hiPSC-derived retinal organoids^[145]7 (Fig. [146]3a).
Quantitative analysis showed mCherry^+ hiPPCs to represent 69.0% ± 6.0
at W1, 87.2% ± 3.14 at W2, and 75.5% ± 4.1 at W3, whereas only 21.9% ±
1.2, 43.9 ± 6.56, and 52.0% ± 17.22 of mCherry^+ cells were observed in
the absence of DAPT at the same corresponding time points
(Fig. [147]3f). RT-qPCR analysis confirmed the upregulation of the
photoreceptor marker CRX in cultures with DAPT at W1, W2, and W3
(Fig. [148]3g). In ProMN culture condition, one million of hiRPCp3
generated 1,81.10^6 ± 0,84, 2,67.10^6 ± 1,09 or 3,58.10^6 ± 0,72
derived retinal cells respectively at W1, W2, and W3. In parallel in
ProMN + DAPT culture condition, one million of hiRPCp3 generated
2.31×10^6 ± 1.12, 2.39×10^6 ± 1,55 or 1.38×10^6 ± 0,64 derived cells
respectively at W1, W2, and W3. Similarly, differentiation during 1
week of the hiRPCp2 or hiRPCp4 generated from the nonfluorescent
hiPSC-2 line showed higher hiPPCs population and CRX expression in the
culture condition using the NOTCH inhibitor DAPT (Supplementary
Fig. [149]10). Quantitative analysis at W1 showed CRX^+ cells to
represent 39.20% ± 4.27 (hiRPCp2) and 37.7% ± 9.32 (hiRPCp4) whereas
only 17.2% ± 12.4 (hiRPCp2) and 23.6 ± 3.37 (hiRPCp4) of hiPPCs were
observed in the absence of DAPT (Supplementary Fig. [150]10). To
highlight the full competence of hiRPCs, we differentiated hiRPCs
during 14 weeks on adherent condition (Supplementary Fig. [151]11). By
immunochemistry, we identified the presence of emergent mature
photoreceptors expressing RHODOPSIN, BLUE OPSIN (OPN1SW) and Red/Green
OPSIN (OPN1MLW), bipolar cells (VSX2/PRKCA) and Müller glia cells
(GS/SOX9). We confirmed by RT-qPCR the expression of the specific genes
CRX, NRL, ARR3, OPN1SW, RHO, AP2, RLBP1, PRKCA targeting these retinal
cell types (Supplementary Fig. [152]11). Concerning the differentiation
of hiRPCs into the RGC lineage, RT-qPCR analysis of BRN3A and BRN3B
expression and immunostaining of BRN3A showed that ProNM and BM can
induce the generation of RGCs from hiRPCs (Fig. [153]3h–j,
Supplementary Figs. [154]9 and [155]10). However, culturing hiRPCs in
BM allowed higher expression of RGC markers at W1 relative to their
culture in ProNM (Fig. [156]3h). Decreased BRN3A and BRN3B expression
after W1 may reflect the loss of hiRPC-derived RGCs under these culture
conditions (Fig. [157]3h). Quantitative analysis of identified RGCs by
immunostaining in BM at W1 showed 7.7% ± 3.5 BRN3A^+ cells among 78.1%
± 5.9 of PAX6^+ cells in the presence of 29.4% ± 3.6 of mCherry^+
hiPPCs (Fig. [158]3i, j).
Fig. 3. Differentiation of hiRPCs into neuroretinal cells.
[159]Fig. 3
[160]Open in a new tab
a Schematic diagram illustrating the differentiation protocol to
generate early retinal cell types. b Immunofluorescence staining and
endogenous expression of differentiated hiRPCp3 after five weeks (W5)
of culture in ProNM for mCherry (hiPPCs), NRL (rod photoreceptor
precursors), AP2 (amacrine cells), LHX1 (horizontal cells), and PAX6
(amacrine and horizontal cells). c High-content analysis of mCherry,
NRL, AP2, and LHX1^+ cells (%) in adherent cultures of hiRPCp3
differentiated in ProNM at W5. Data are presented as mean ± SD (n = 6
for mCherry and n = 12 for NRL, AP2 and LHX1). d RT-qPCR analysis of
neuroretinal markers (CRX, NRL, AP2, and LHX1) in hiRPCp3
differentiated in ProNM from W1 to W3 and at W5. Data are normalized to
that of hiRPCp3 at W1 and presented as mean ± SD (n = 3 per time
point). e Endogenous expression of mCherry of hiRPCp3 differentiated in
ProNM ± DAPT from W1 to W3. f High-content analysis of mCherry^+ cells
(%) in adherent cultures of hiRPCp3 differentiated in ProNM ± DAPT from
W1 to W3. Data are presented as mean ± SD (n = 8 per time point). g
RT-qPCR analysis of the PPC marker CRX in hiRPCp3 differentiated in
ProNM ± DAPT from W1 to W3. Data were normalized to that of hiRPCp3
differentiated in ProNM at W1 and presented as mean ± SD (n = 3 per
time point). h RT-qPCR analysis of the retinal ganglion cell (RGC)
markers BRN3A and BRN3B in hiRPCp3 differentiated in ProNM or BM from
W1 to W3 and W5. Data were normalized to that of hiRPCp3 differentiated
in ProNM at W1 and presented as mean ± SD (n = 3 per time point). i
Immunofluorescence staining of hiRPCp3 differentiated in BM at W1 for
mCherry (hiPPCs), BRN3A (RGCs), and PAX6. j High-content analysis of
mCherry (hiPPCs), BRN3A (RGCs), and PAX6^+ cells (%) in adherent
cultures of hiRPCp3 differentiated in BM at W1. Data are presented as
mean ± SD (n = 5 for mCherry and BRN3A and n = 6 for PAX6). One-way
ANOVA followed by a Dunnett’s multiple comparison test (d, h).
Comparison to W1 (d) or W1 ProNM (h). Two-tailed Student’s t-test for
two-group comparisons (g). ****p < 0,0001; ***p < 0.001;
**p < 0.01; *p < 0.05. Nuclei were counterstained with DAPI (blue).
hiPSC-5FC-derived cells. Scale bar: b, e, i, 50 µm.
Retinal pigmented epithelial cell generation from hiRPCs
Based on the identification of the preneurogenic state of hiRPCs, we
tested the ability of these new progenitors to differentiate into RPE
cells. hiRPCs were submitted to spontaneous differentiation using BM
for 12 weeks (W12) (Fig. [161]4a). Under these conditions, pigmented
cells could be observed (Fig. [162]4b) and phase contrast imaging
confirmed the RPE cell morphology (Fig. [163]4c). RT-qPCR analysis for
specific RPE markers showed increased expression of MITF, PEDF, VEGFA,
and BEST1 from W1 to W12, confirming cell commitment towards the RPE
lineage (Fig. [164]4d). To exclude the potential contamination of RPE
cells in banked hiRPCs, we added FGF2, reported to be a factor
necessary for the differentiation of retinal progenitors towards the
neuroretinal lineage, at the expense of the RPE lineage, during
retinogenesis^[165]23. The presence of FGF2 completely prevented the
appearance of RPE cells and markers (Fig. [166]4b, d, e). HiRPC-derived
RPE (hiRPE) cells at W12 were passed (noted as hiRPEp0) and formed a
confluent cell monolayer after 1 week that displayed the classical RPE
morphology (Fig. [167]4f). Immunostaining targeting MITF and ZO-1
confirmed the identity of RPE cells, with regular apical tight
junctions (Fig. [168]4g, h) and without mCherry expression
(Fig. [169]4i).
Fig. 4. Differentiation of hiRPCs into RPE cells.
[170]Fig. 4
[171]Open in a new tab
a Schematic diagram illustrating the differentiation protocol of RPE
generation from hiRPCp3. b Photograph of a six-well plate of
differentiated hiRPCp3 at W12 using BM ± FGF2. c Phase-contrast image
of hiRPCp3 cultured in BM at W12. d RT-qPCR analysis of specific RPE
markers in hiRPCp3 cultured in BM or BM + FGF2 from W1 to W3 and at W5
and W12. All expression was normalized to that of W1 hiRPCs cultured in
BM. Data are presented as mean ± SD (n = 3 per time point). e
Phase-contrast image of hiRPCp3 at W12 cultured in BM + FGF2. f
Phase-contrast image of hiRPC-derived RPE at passage 0 (hiRPEp0) at W1.
g immunofluorescence staining of hiRPEp0 for MITF, ZO1 at W1. h Z-stack
confocal image of the apical ZO1 marker on hiRPEp0 at W1. i Endogenous
mCherry staining in hiRPEp0 at W1. One-way ANOVA followed by a
Dunnett’s multiple comparison test (c, e, g, h). Comparison to W1.
****p < 0,0001; ***p < 0.001; **p < 0.01; *p < 0.05. Nuclei were
counterstained with DAPI (blue). hiPSC-5FC-derived cells. Scale bar: c,
e, g, I, 50 µm. h, 10 µm.
Discussion
In this study, we demonstrate that RPCs within hiPSC-derived ROs can be
isolated and cryo-preserved after steps of expansion in adherent
culture conditions. Banked hiRPCs maintained their multipotency
illustrated by their ability to differentiate into all retinal cell
types. The multipotency of the hiRPCs was maintained by culture
conditions using a RPC-dedicated medium containing specific factors to
mimic the environment of retinogenesis. During retinal development,
RPCs have to maintain the proper balance between proliferation and
differentiation to produce the full range of retinal cell types in
sufficient numbers to generate a complete retina^[172]24. It is well
established that cell fate determination is controlled by a combination
of extrinsic cues and intrinsic factors^[173]24–[174]26. Based on these
data, various approaches, focusing mainly on extracellular signals,
have been developed to isolate multipotent RPCs and amplify them.
However, these processes failed to maintain multipotency in expanded
RPCs^[175]12–[176]14. Based on the literature, we hypothesized that
modulating specific signaling pathways, such as WNT, SHH, EGF, and FGF
signaling, could be sufficient to act on the intrinsic factors that
control the proliferation and multipotency of RPCs. To recapitulate the
environment of the developing tissue, we selected five extrinsic
factors: the GSK3 inhibitor CHIR99021, mimicking WNT activation through
beta-catenin accumulation in the cytoplasm and maintaining the human
stem cell fate and expression of retinal progenitor
markers;^[177]27,[178]28 PURMORPHAMINE as an activator of the Shh
pathway, sustaining RPC self-renewal and
multiplication^[179]26,[180]29–[181]33 and reproducing Shh pathway
activation by ganglion cells (RGCs) during retinogenesis; FGF2, the
most highly used factor to maintain the pluripotency of human embryonic
stem cells and iPSCs in culture and shown to also potentiate the action
of the Shh pathway and avoid RPE differentiation of
progenitors^[182]34,[183]35; EGF, classically coupled to FGF2 in neural
stem cell culture and promoting RPC proliferation at the expense of
their differentiation^[184]36; and ATP, which controls the cell cycle
and RPC proliferation in the developing retina^[185]37–[186]40,
mimicking RPE-release of ATP into the subretinal space^[187]37. Thus,
we demonstrate that simultaneous activation of these specific pathways
allows the selection and amplification of hiRPCs. Transcriptomic
analysis (RT-qPCR and RNAseq) and immunofluorescence staining confirmed
the high similarity between RPCs within ROs and hiRPCs thawed and
expanded until passage 4. The proneurogenic phenotype of hiRPCs was
highlighted by transcriptomic analysis, as well as by the
differentiation capacity of the hiRPCs to differentiate into all neural
retinal cell types including mature photoreceptors. However, although
at W14 few differentiated photoreceptors expressed mature markers as
RHODOPSIN, BLUE OPSIN or RED/GREEN OPSIN, these expressions were
consistent with the retinogenesis described in ROs at this
time^[188]16. Interestingly, in addition to the ability to
differentiate into neuroretinal cells and muller glial cells, hiRPCs
can also be differentiated into RPE cells. This shows that RPCs within
ROs still possess the intrinsic ability to regain the preneurogenic
phenotype, depending on the extrinsic environment. This valuable
multipotent cell line was easy to use and produced millions of
differentiated retinal cells in a short time under adherent conditions
from a cryopreserved cell stock. From one banked tube of hiRPCp1, more
than 10 millions of hiPPCs can be produced within enriched cultures of
up to 90% in 4 weeks and without a purification step. This scale-up
production of hiPPCs should be useful for disease modeling, tissue
engineering and drug discovery (Fig. [189]5). This potential source of
transplantation-compatible cell population^[190]41–[191]43 was produced
under xeno-free conditions as needed for future cell therapies but the
in vivo maturation capabilities of the hiPPCs still need to be
evaluated at this stage. Moreover, we showed differences in the
differentiation ability among lines. This observation could indicate
that the treatment with molecules, as the NOTCH inhibitor DAPT, must to
be adapted to obtain optimal number of expected cells. Similarly, the
ability to generate RGCs from hiRPCs may pave the way towards the
future treatment of diseases that affect the optical nerve, such as
glaucoma or diabetic retinopathy. In addition to being a substrate for
the scaling up of the production of retinal cell types, hiRPCs appear
to be a useful tool to study retinogenesis and associated diseases.
Indeed, transcriptomic analysis comparing RO and hiRPC gene expression
revealed potential markers of multipotency, neurogenesis, and
differentiation. Of note, specific transcriptomic analysis using gene
expression filters selected genes involved in developmental retinal
diseases, such as microphthalmia (VAX1^[192]44, ALDH1A3^[193]45,
GJA1^[194]46, SMOC1^[195]47) and retinal dystrophies, such as
age-related macular dystrophy (ADAMTS9^[196]48,[197]49), Leber
congenital amaurosis (KCNJ13^[198]50,[199]51), glaucoma (CA2^[200]52),
and Stargardt disease (ABCA4^[201]53), supporting the use of this
workflow to identify new genes that cause retinal diseases. Overall, we
used an innovative culture condition to generate a human retinal
multipotent cell line. Although future experiments are needed to
evaluate all applications using hiRPCs and its derivatives, this
advanced cell line could become a gold standard cellular substrate to
study retinogenesis, create disease models, and generate retinal cell
libraries useful for cell therapy and drug discovery (Fig. [202]5).
Thus, bankable hiRPCs could convert hope to reality for the future
accessibility of innovative treatments for millions of people suffering
from degenerative retinal diseases.
Fig. 5. Usefulness of hiRPCs and their cell derivatives.
[203]Fig. 5
[204]Open in a new tab
hiRPCs could be used for retinogenesis studies, developmental disease
modeling, the generation of retinal cell libraries, and as cell
substrates for cell engineering. Derived retinal cells from hiRPCs
could be used for patient-based degenerative disease modeling, drug
discovery, cell therapy, and tissue engineering.
Methods
Human subjects
Postmortem eye tissues used to generate the hiPSC-5F^[205]17 and
hiPSC-5FC^[206]15 clones were collected in accordance with the French
bioethics law at the Laboratory of Anatomy of the Faculty of Medicine
of St-Etienne, France. Handling of donor tissues adhered to the tenets
of the Declaration of Helsinki of 1975 and its 1983 revision in
protecting donor confidentiality. Skin biopsy used to generate the
hiPSC-2 clone^[207]7,[208]16 were obtained from informed patient under
the approval of French regulatory agencies.
Human iPSC culture
Experiments were conducted using the human fluorescent reporter
AAVS1::CrxP_H2BmCherry-hiPSC line (hiPSC-5FC line), allowing the
identification of photoreceptor lineage-committed cells by endogenous
mCherry staining^[209]15 and with the nonfluorescent hiPSC-5F^[210]17
and the hiPSC-2 lines^[211]7,[212]16. HiPSCs were cultured under
feeder-free conditions on truncated recombinant human vitronectin
rhVTN-N (STEMCELL Technologies)-coated dishes in mTeSR^TM1 Medium
(STEMCELL Technologies). Cells were routinely cultured in 6-cm^2 dishes
at 37 °C in a standard 5% CO[2]/95% air incubator with a daily medium
change. HiPSCs were passaged weekly, as previously described, using
2 mL enzyme-free gentle cell-dissociation reagent (STEMCELL
Technologies) for 6 min at room temperature^[213]16. For
immunostaining, hiPSCs were passaged and replated in 24 well-plates
containing glass coverslips precoated with human vitronectin rhVTN-N
(STEMCELL Technologies). Cells were returned to the incubator at 37 °C
and 5% C02 for 1 week and then fixed with 4% PFA in PBS for 10 min.
Retinal organoid differentiation
RO generation was based on our previously established adherent hiPSC
differentiation protocol^[214]7,[215]16,[216]18. HiPSCs were expanded
to 70 to 80% confluence in 6-cm diameter dishes as described above. At
this time, defined as day 0 (D0), hiPSCs were cultured in chemically
defined Essential 6 (E6) medium with 10 units/ml penicillin and
10 mg/ml streptomycin (Thermo Fisher Scientific). At D2, cells were
switched to E6N2 medium, composed of E6 medium, 1% N2 supplement, 10
units/ml penicillin, and 10 mg/ml streptomycin (Thermo Fisher
Scientific). Media was changed every 2 to 3 days. After 4 weeks (W4),
identified self-formed retinal organoids were isolated using a needle
and cultured in six-well-plates (10–15 organoids per well) as floating
structures in proneural medium (ProNM) supplemented with 10 ng/ml
animal-free recombinant human FGF2 (Peprotech) and half of the medium
was changed every 2 to 3 days. ProNM is composed of chemically defined
DMEM:Nutrient Mixture F-12 (DMEM/F12, 1:1, L-Glutamine), 1% MEM
nonessential amino acids, 2% B27 supplement (Thermo Fisher Scientific),
10 units/ml penicillin, and 10 mg/ml streptomycin. After 1 week, FGF2
was removed from the ProNM and half of the medium was changed every 2-
to 3 days.
Isolation, expansion, and cryopreservation of hiRPCs
HiRPCs were isolated from 6-week-old (W6) ROs. Before dissociation, the
surrounding pigmented portion of the ROs was discarded under a
stereomicroscope. Between 70 to 90 structures were washed twice in
Ringer’s solution (155 mM NaCl, 5 mM KCl, 2 mM CaCl[2], 1 mM MgCl[2],
2 mM NaH[2]PO[4], 10 mM HEPES, and 10 mM glucose) and enzymatically
dissociated using two units of papain (Worthington, WOLS3126),
previously activated in SAP solution (125 mM NaCl, 3.6 mM KCl, 1.18 mM
MgCl[2], 22.6 mM NaHCO[3], 0.02 mM NaH[2]PO[4], 0.028 mM Na[2]HPO[4],
1.2 mM Na[2]SO[4], 10 mM glucose; 0.54 mM Na[2]EDTA), for 30 min at
37 °C. ROs were dissociated by up and down pipetting in the presence of
1 μg/ml DNAse (Sigma-Aldrich), preventing cell aggregation. After
complete dissociation, the papain was inactivated with prewarmed ProNM.
Cells were centrifuged and resuspended in prewarmed RPCM medium. RPCM
is composed of E6 medium, 10 units/ml penicillin and 10 mg/ml
streptomycin (Thermo Fisher Scientific), 3 µM CHIR99021 (Euromedex),
1 µM Purmorphamine (Euromedex), 10 ng/mL FGF2 (Peprotech), 100 ng/mL
EGF (Peprotech), and 100 µM ATP (Sigma-Aldrich). Cells were plated at a
density of 3 ×10^5 cells/cm² onto T25-cm^2 dishes previously coated
with Geltrex^TM (Thermo Fisher Scientific). At this time, hiRPCs were
designated as being at passage 0 (hiRPCp0). Cells were incubated at
37 °C in a standard 5% CO[2]/95% air incubator and the medium was
changed every 2 to 3 days for 1 week. At 70 to 80% confluency, hiRPCp0
cultured in RPCM were dissociated using 2 mL TrypLE^TM Express (Gibco)
per T25-cm^2 for 8 min at 37 °C and the reaction stopped by dilution
with 8 mL prewarmed RPCM. HiRPCs were centrifuged at 110 × g for 3 min
and resuspended in prewarmed RPCM. Cell proliferation was measured
using an automated cell counter (Scepter 3.0 Handheld Automated Cell
Counter, Millipore). Cells were seeded in Geltrex^TM (Thermo Fisher
Scientific) precoated T25-cm^2 plates at a density of 1.5 ×10^5
cells/cm² and designated as hiRPCs at passage 1 (hiRPCp1). Cells were
incubated at 37 °C in a standard 5% CO[2]/95% air incubator and the
medium was changed every 2 to 3 days for 1 week. At 70%–80% confluency,
hiRPCp1 were dissociated using TrypLE^TM Express (Gibco) and
cryopreserved at 5 ×10^6 cells/ml using Cryostor® cryopreservation
medium (STEMCELL Technologies) in cryogenic tubes. Tubes were placed in
an isopropanol-based freezing container at −80 °C for a minimum of 4 h
and kept in a −150 °C freezer for long-term storage.
Molecular cocktail testing
Experiments were conducted using derived cells from the human
fluorescent reporter hiPSC-5FC line and the hiPSC-2 line. After
complete ROs dissociation, cells were centrifuged and resuspended in 6
different culture conditions: E6 medium; E6 medium and 1 µM
Purmorphamine; E6 medium and 3 µM CHIR99021; E6 medium and 100 µM ATP;
E6 medium, 1 µM Purmorphamine, 3 µM CHIR99021, 100 µM ATP or RPCM
medium. For immunostaining, hiRPCp0 were replated at a density of 3
×10^5 cells/cm² in 24 well-plates containing glass coverslips with a
glass bottom (Celvis) precoated with Geltrex^TM (Thermo Fisher
Scientific). Cells were returned to the incubator at 37 °C and 5% C02
for 1 week and then fixed with 4% PFA in PBS for 10 min.
Differentiation of hiRPCs
The majority of differentiation experiments were performed using
hiRPCp3 from banked hiRPCp1. Thawed hiRPCp1 were seeded at 5 ×10^4
cells/cm² (noted hiRPCp2) using Geltrex® (Thermo Fisher Scientific)
precoated T25-cm^2 dishes or six-well plates. After 1 week under
expansion conditions using RPCM, hiRPCp2 were passaged and experiments
performed using 1-week expanded hiRPCp3. Spontaneous differentiation
was performed using a basal medium (BM) composed of E6 medium, 10
units/ml penicillin, and 10 mg/ml streptomycin (Thermo Fisher
Scientific). Directed differentiation was performed using ProNM (i.e.,
retinal differentiation). Cell culture to promote hiPPCs was performed
using ProNM supplemented with the Notch Inhibitor DAPT at 10 μM from
day 2 to day 7 of the 3 weeks of differentiation. For immunostaining
and high-content image quantification at the end of differentiation,
hiRPC-derived retinal cells were enzymatically dissociated using 0.24
units/cm^2 papain as previously described^[217]16. Cells were replated
at 1.5 ×10^5 cells/cm^2 in 24 well-plates containing glass coverslips
or in 96-well plates with a glass bottom (Celvis) precoated with
Poly-D-lysine (2 µg/cm², Merk) and laminin (1 µg/cm², Sigma-Aldrich).
Cells were returned to the incubator at 37 °C and 5% C0^2 for 24 h and
then fixed with 4% PFA in PBS for 10 min. For long-term differentiation
experiments, hiRPCp2 (hiPSC-5F line) were cultured in ProNM during 14
weeks supplemented with FGF2 in the first 2 weeks. At W7, hiRPC-derived
retinal cells were enzymatically dissociated using papain as previously
described and replated at 1.5 ×10^5 cells/cm^2 in 6-well plates
precoated with Poly-D-lysine (2 µg/cm², Merk) and laminin (1 µg/cm²,
Sigma-Aldrich) allowing the culture differentiation up to W14. Cells
were incubated at 37 °C in a standard 5% CO[2]/95% air incubator and
the medium was changed every 2 to 3 days.
RNA extraction and Taqman assay
Total RNA was extracted using a Nucleospin RNA II kit (Macherey-Nagel)
according to the manufacturer’s protocol and RNA yields and quality
assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific).
cDNA was synthesized from 100 ng total RNA using the QuantiTect reverse
transcription kit (Qiagen) following the manufacturer’s
recommendations. Synthesized cDNA was then diluted 1/20 in DNase-free
water before performing real-time quantitative PCR (RT-qPCR). RT-qPCR
analysis was performed using an Applied Biosystems real-time PCR device
(7500 Fast System) with custom TaqMan® Array 96-Well Fast plates and
TaqMan® Gene expression Master Mix (Life Technologies) following the
manufacturer’s instructions. All primers and MGB probes labeled with
FAM for amplification were purchased from Life Technologies
(Supplementary Data [218]1). Results were normalized against those
obtained with 18 S rRNA and the quantification of gene expression was
based on the delta-deltaCt method (Eq. [219]1) in three minimum
independent biological experiments.
[MATH: 2−ddCtwithddCt=[(Ctgene1−Ct18S)t2]n−[(Ctgene1−Ct18S)t1]n :MATH]
1
where Ct = cycle threshold; t = time; n = independent biological
experiments.
RNAseq analysis
RNA-seq libraries were constructed by Integragen Genomics® from 400 ng
total RNA (RIN > 7) using NEBNext Ultra II Directional RNA (New England
Biolabs) and paired-end sequencing of 100 base-pair fragments was
performed on a Novaseq 6000 system (Illumina). Image analysis and base
calling was performed using an Illumina Real Time Analysis (3.4.4)
device with default parameters. Pass-filtered reads were mapped using
STAR 2.7.3a^[220]54 and aligned to Ensembl genome assembly GRCh38
(release 98). This annotation includes cDNA, miRNA, long noncoding RNA,
pseudogene, and gene predictions. For differential expression analysis,
a count table of the gene features was obtained using
FeatureCounts^[221]55. For gene level analysis, EdgeR was used for
normalization, differential expression analysis, and to compute TPM
(transcripts per million) values^[222]56. Comprehensive gene-list
analysis, enriched biological pathways, and gene annotation from
differential expression were based on the Gene Ontology (GO)
classification system using Metascape^[223]57. R packages were used for
data mining, including GOPlot for pathway data graphical
representation^[224]58. GEO accession: [225]GSE220792.
High-content Image analysis
For high-content quantitative analysis, images were acquired using an
automated microscope (Arrayscan VTI HCS Reader, Thermo Fisher
Scientific) of the screening facility. To count the number of hiPPCs
expressing mCherry (red) or immunofluorescence-stained cells, an image
analysis workflow was created using HCS Studio Cell Analysis Software
(Thermo Fisher Scientific) with the TargetActivation BioApplication.
Image analysis included an image preprocessing step followed by a
segmentation step, allowing classification of the pixels and objects
based on fluorescence intensity thresholds. High-content quantitative
analysis of hiPPCs derived from hiPSC-2 line were acquired using the
automated microscope CQ1 (Confocal Quantitative Image Cytometer,
Yokogawa) » and analyze in by the CellpathFinder Software (Yokogawa).
Immunostaining of ROs, hiRPCs, and hiRPC-derived cells
RO sections or retinal cells were fixed with 4% PFA in PBS for 10 min
before immunostaining. After washing with PBS, nonspecific binding
sites were blocked for 1 h at room temperature with PBS containing 0.2%
gelatin and 0.25% Triton X-100 (blocking buffer) and then overnight at
4 °C with the primary antibody (Supplementary Data [226]2) diluted in
blocking buffer. Samples were washed four times for 5 min in PBS with
0.1% Tween and then incubated for 1 h at room temperature with either
AlexaFluor 488 or 647 secondary antibodies (Interchim) diluted at 1:600
in blocking buffer. Cells were washed two times for 5 min in PBS with
0.1% Tween and once for 5 min in PBS with 0.1% Tween and DAPI
(4’,6-diamidino-2-phenylindole) at 1:1000. Samples were washed with PBS
and mounted on slides for imaging. Fluorescence was captured using an
Olympus FV1000 confocal microscope equipped with 405, 488, 543, and
633 nm lasers. Images were acquired using a 1.55 or 0.46 µm step size
and corresponded to the projection of 20 to 40 optical sections.
Statistics and reproducibility
Statistical analyses represent the mean of at least three independent
experiments. Data were averaged and are expressed as means ± SDs
(Standard Deviation scores). Statistical analysis was performed using
Prism 9 (GraphPad software) with appropriate statistical tests. A
two-tailed Student’s t-test was carried out for two-group comparisons,
and ordinary one-way analysis of variance (ANOVA) followed by a
Dunnett’s test was performed for multiple-group comparisons. Values of
p < 0.05 were considered statistically significant.
Supplementary information
[227]Editorial Assessment Report^ (192.8KB, pdf)
[228]Supplementary Information^ (9.7MB, pdf)
[229]Supplementary Data 1^ (15.6KB, docx)
[230]Supplementary Data 2^ (17.6KB, docx)
[231]Supplementary Data 3^ (9MB, xlsx)
[232]Supplementary Data 4^ (6.8MB, xlsx)
[233]Supplementary Data 5^ (423.5KB, xlsx)
[234]Supplementary Data 6^ (49.7KB, xlsx)
Acknowledgements