Abstract
Current approaches in myocardial infarction treatment are limited by
low cellular oxidative stress resistance, reducing the long-term
survival of therapeutic cells. Here we develop a liquid-crystal
substrate with unique surface properties and mechanical responsiveness
to produce size-controllable cardiospheres that undergo pyroptosis to
improve cellular bioactivities and resistance to oxidative stress. We
perform RNA sequencing and study cell metabolism to reveal increased
metabolic levels and improved mitochondrial function in the
preconditioned cardiospheres. We test therapeutic outcomes in a rat
model of myocardial infarction to show that cardiospheres improve
long-term cardiac function, promote angiogenesis and reduce cardiac
remodeling during the 3-month observation. Overall, this study presents
a promising and effective system for preparing a large quantity of
functional cardiospheres, showcasing potential for clinical
application.
Subject terms: Regeneration, Tissue engineering, Biomedical
engineering, Cardiovascular diseases
__________________________________________________________________
Therapeutic options for myocardial infarction therapy remain limited.
Here the authors report the application of an optimized liquid crystal
substrate in the mass production and effective preconditioning of
cardiospheres, which could generate cardiospheres with improved cell
bioactivity and resistance to oxidative stress for myocardial
infarction therapy.
Introduction
The 30-day mortality following myocardial infarction (MI) was 13.6% on
average^[44]1. When MI occurs, myocardial ischemia causes a series of
irreversible pathological processes, such as severe inflammation,
massive cell death, and cardiac fibrosis, which ultimately lead to
heart failure^[45]2,[46]3. To date, many clinical and animal studies
have shown cell-based therapies as promising approaches to reverse or
slow MI disease progression^[47]4–[48]7.
To pursue satisfactory therapeutic outcomes, many effective cell
processing methods have been extensively developed to improve cell
bioactivities. Hanging drops, spinner flasks, and three-dimensional
(3D) bioprinting have been used to prepare spheroids or
organoids^[49]8–[50]10. By providing mechanical cues, extracellular
matrix (ECM), and soluble factors in native niches, the 3D spheroids
could promote pluripotency marker expression (Nanog, Oct4, Sox2),
cardiac lineage differentiation, paracrine secretion,
anti-inflammation, and antisenescence^[51]11–[52]14. To further improve
cell survival in hostile environments and their therapeutic potential,
many researchers have suggested that simulating the inflammatory
environment with preconditioning strategies could enhance cell
resistance to adverse effects. Hypoxia and low-concentration
inflammatory factor treatment are widely used preconditioning
strategies^[53]15,[54]16. Following preconditioning treatment, the
phenotype of pretreated cells shifted in therapeutically desirable
directions, and their abilities to resist inflammation were greatly
enhanced^[55]17–[56]19. These studies demonstrated the feasibility and
effectiveness of preconditioning treatment, and they highlighted the
significance of enhancing cellular bioactivities and inflammation
resistance in damaged tissue regeneration.
Cardiosphere-derived cells (CDCs) are of endogenous cardiac
origin^[57]20 and possess the ability to form 3D spherical clones,
cardiospheres (CSps), in vitro^[58]21. Compared to monolayer CDCs, CSps
possessed improved growth factor secretion and cardiac regeneration
potential, making them a good cell source for MI therapy^[59]22,[60]23.
Previous reports demonstrated that CSps could regulate the inflammation
of infarcted myocardium through immunomodulatory effects^[61]24–[62]26,
and it was proposed that the CDCs polarized macrophages away from the
M1 phenotype but not toward a classical M2 state, but to a distinct
cardioprotective phenotype that promotes the survival of ischemic
cardiomyocytes^[63]27. Also, the secretion of various growth factors
and bioactive molecules from CSps could involve in the vessel network
rebuilding process, which were beneficial for reducing ventricular
adverse remodeling and hypertrophy^[64]28–[65]31. These characteristics
make CSps a good cell source for MI therapy. Previously,
preconditioning CSps with pericardial fluid obtained from myocardial
infarction was prepared by our colleagues Zhang et al., and the
paracrine function and survival rate of the pericardial
fluid-pretreated CSps dramatically increased, exhibiting significant
improvement of MI cardiac function, and the DiR-labeled CSps showed
cTnT-positive in the infarcted area, indicating the direct
differentiation of CSps into cardiomyocytes^[66]32. Moreover, Zhang et
al. reported that pericardial application could serve as a new and
effective route for CSps transplantation, and this therapeutic strategy
also showed favorable potential for further clinical
application^[67]33.
Cellular physiological activities could be manipulated by the
properties of the contacted substrate^[68]34–[69]36. Liquid-crystal
patterns could directly introduce cells into a 3D environment and form
cell structures in situ^[70]37, which may be beneficial for mass
spheroid production during large-scale clinical applications. In
addition, the alignment of monolayer support cells could form a nematic
liquid crystal pattern to induce cell death at the stress
localization^[71]38. Pyroptosis is an inflammation-related cell death
program, and the pyroptotic cells could release complex inflammatory
signals to affect surrounding cells^[72]39. Considering the
inflammatory signals released from the injured cells would be more
efficient in improving the cytoprotective function of the target cells
than the artificial stimulus inducer^[73]15, the phenomenon of liquid
crystal pattern to induce cell death might be applied to develop a
local dynamic inflammatory milieu and turn this theoretical model into
cell product preparation methods. Therefore, using a liquid crystal
substrate for pretreated CSps might be a stable, convenient, and
effective strategy to achieve mass production and cell function
improvement.
In this work, a comprehensive optimized 3D culture platform for
effective CSps production and preconditioning are developed using a new
kind of cholesteric liquid crystal substrate, octyl hydroxypropyl
cellulose ester (OPC)^[74]40. The OPC substrate could promote 3D
spheroid formation and induce cell death with its unique properties of
liquid crystals, and the internal cells in the spheroid could be
activated by inflammatory factors secreted from the external cells. The
bioactivities, metabolism, and function of OPC-CSps are analyzed, and
their therapeutic effects on heart function, angiogenesis, inflammatory
infiltration, and ventricular remodeling are evaluated in a rat MI
model.
Results
Liquid-crystal OPC possessed mechanical responsiveness
The synthesis schematic diagram of OPC is shown in Fig. [75]1a. The
polarized light microscopic images revealed that the OPC displayed the
characteristics of liquid crystals, including birefringence, fissures,
and fingerprint-like texture (Fig. [76]1b). The atomic force microscope
results showed that the surface of the OPC substrate was a nonflat
profile with wavy bulges. The height of the grains ranged from
20–35 nm, and the roughness (root mean square height, Sq) was
2.33 ± 0.29 nm (Fig. [77]1c). The static contact angle of the OPC
substrate was 106.49 ± 2.36°, indicating that it had a hydrophobic
surface (Fig. [78]1d). The effect of shear force on the OPC substrate
surface was examined. As the X-ray diffraction (XRD) results showed,
there were two diffraction peaks before shearing, and peaks at
approximately 2θ of 20–22° were enhanced following the application of
shear force, indicating the rearrangement of the liquid crystal unit
(Fig. [79]1e). In addition, the average crystallization rate and the
grain size of the vertical (002) crystal plane were calculated
according to the XRD results, and the average crystallization rate
increased from 17.91% to 21.48%, and the grain size of the vertical
(002) crystal plane increased from 0.69 nm to 1.14 nm. The
viscoelasticity of the OPC substrate was examined by a
stress-controlled rheometer, and the phase transition was observed. At
1–10 rad, the loss modulus (G″) was higher than the energy storage
modulus (G′), and OPC preferred viscous deformation behavior. In
contrast, at 10–100 rad, the energy storage modulus (G′) was higher
than the loss modulus (G″), indicating an elastic deformation tendency
(Fig. [80]1f). The OPC substrate was nontoxic in cell culture
(Fig. [81]1g).
Fig. 1. OPC showed liquid crystal characteristics and mechanical
responsiveness.
[82]Fig. 1
[83]Open in a new tab
a Schematic diagram of OPC synthesis. b Representative image of OPC
under polarized light microscopy, and 10 independent samples were
observed. c Representative atomic force microscopy results of OPC
(n = 5). d The static contact angle (θ) of OPC (n = 7). e The XRD
results of OPC before and after shear force application. f The storage
modulus (G′) and loss modulus(G″) over 1–100 angular frequency measured
by a stress-controlled rheometer. g The results of the OPC toxicology
test (n = 5 biologically independent samples). All data are shown as
the mean ± SD, two-way ANOVA (g).
The OPC substrate promoted CSps formation and progenitor phenotypes
The formation of CSps on the polystyrene (PS) substrate, the ultralow
attachment (ULA) substrate and the OPC substrate was observed, and
different shapes of CSps were acquired (Fig. [84]2a and Supplementary
Fig. [85]1). The adherent CDCs on the PS substrate converged and formed
regular spherical aggregates, and supporting cells surrounded at the
base of the PS-CSps. The ULA-CSps were formed by suspended single-cell
stacking, and they developed into noncircular, oval, and irregular
shapes. On the OPC substrate, CDCs initially attached to the substrate
and gradually aggregated to form regular and circular spheroids, and no
supporting cells were observed around the OPC-CSps. During the
formation of CSps, the sizes of OPC-CSps showed a stable growth
tendency compared with the obvious increase of the PS-CSps and the
ULA-CSps (Fig. [86]2b and Supplementary Fig. [87]1). Following a 3-day
cultivation, the sizes of PS-CSps, ULA-CSps, and OPC-CSps were
280.96 ± 40.56 μm, 203.34 ± 69.36 μm, and 120.29 ± 15.34 μm,
respectively. In addition, the spheroid density on the OPC substrate
was significantly higher than that on the other two substrates
(Fig. [88]2c).
Fig. 2. OPC-CSps displayed controllable spheroid size and favorable
progenitor cell phenotypes.
[89]Fig. 2
[90]Open in a new tab
a Morphological change in CSps on the PS, ULA, and OPC substrates. b
Quantification results of the spheroid size in 5-day cultivation on the
PS, ULA, and OPC substrates (n = 30 images from 6 independent
experiments). c Quantification results of the CSps density in 5-day
cultivation on each substrate (n = 8 from 4 independent experiments). d
Phenotype characterization of CSps from each group after 3 days of
cultivation. The proportions of positive cells relative to the isotype
control are shown (n = 3 biologically independent samples). All data
are shown as the mean ± SD, *P < 0.05 vs. PS, ^#P < 0.05 vs. ULA.
One-way ANOVA (d) or two-way ANOVA (b, c).
The expression levels of CSps surface markers were analyzed. Compared
to the PS group (KDR:3.15 ± 0.85%, Sca-1: 12.61 ± 3.63%) and the ULA
group (KDR: 3.76% ± 0.79%, Sca-1: 11.13 ± 2.39%), the OPC group
exhibited the highest expression of KDR (8.55 ± 1.20%) and
Sca-1(26.17 ± 4.17%) (P < 0.05). In addition, the ULA group
(CD31:11.74 ± 0.89%, CD34: 13.91 ± 1.64%) and the OPC group (CD31:
11.99 ± 1.07%, CD34: 14.47 ± 0.85%) showed an increase in the
expression of CD31 and CD34 when compared to the PS group (CD31:
2.68 ± 1.24%, CD34: 1.80 ± 0.55%) (P < 0.05), along with a decrease in
CD90 (PS: 98.47 ± 0.65%, ULA: 68.37 ± 4.81%, OPC: 6.87 ± 1.01%)
(P < 0.05) and no significant difference in CD105 compared to the PS
group (PS: 45.73 ± 7.03%, ULA: 37.03 ± 3.50%, OPC: 39.53 ± 8.51%)
(Fig. [91]2d).
OPC-induced pyroptosis improved CSps cellular bioactivities and paracrine
effects
The expression of caspase-1 was observed in the peripheral cells of the
OPC-CSps, while no positive signals were observed in the PS group and
the ULA group (Fig. [92]3a). The transcription levels and protein
expression levels of the pyroptosis-related key factors caspase-1 and
IL-1β in the OPC group were both significantly upregulated compared
with those in the PS group and the ULA group (P < 0.05)
(Fig. [93]3b–d). The transmission electron microscope (TEM) analysis
was performed to evaluate the cellular ultrastructure. Highly cell
aggregation was observed in the ULA-CSps, while there was certain
cell-cell space remained in the center of the OPC-CSps (Supplementary
Fig. [94]2). Normal structures of the nucleus, mitochondria, and rough
endoplasmic reticulum were observed in the PS group. However,
endoplasmic reticulum dilatation, degranulation, and swollen
mitochondria were observed in the cells from the ULA group. Moreover,
cells in the OPC-CSps showed normal nuclear structures and abundant
normal mitochondria. In contrast to the PS group and the ULA group,
many microvesicles could be observed on the membrane surface
(Fig. [95]3e). Following a 3-day cultivation, the cell survival rate of
the ULA group was markedly lower than that of the PS group. The cell
survival rate of the OPC group was significantly higher than that of
the ULA group (P < 0.05), and it showed no significant difference from
the PS group (Fig. [96]3f). The expression of Ki67 was observed in the
center of the PS-CSps and the OPC-CSps, while the Ki67^+ cells were
mostly found in the periphery of the ULA-CSps (Supplementary
Fig. [97]3). Additionally, the proliferation ability of the CDCs from
OPC-CSps was significantly higher in the OPC-CSps than that from
ULA-CSps (P < 0.05), and it showed no significant difference from the
PS-CSps (Fig. [98]3g). In addition, the transcription levels of the
cell pluripotency markers Oct4, Nanog, and Sox2 (Fig. [99]3h) and the
paracrine-related genes VEGF, HGF, IGF-1, and bFGF dramatically
increased following OPC culture compared with the PS and ULA groups
(P < 0.05) (Fig. [100]3i).
Fig. 3. OPC-induced pyroptosis improved CSps cellular bioactivities and
paracrine effects.
[101]Fig. 3
[102]Open in a new tab
a Representative image of caspase-1 immunofluorescence staining results
of each group. b The mRNA transcription levels of caspase-1 and IL-1β
(n = 5 biologically independent samples). c The protein expression
levels of caspase-1 and cl-caspase-1 tested by western blot. d The
concentration of IL-1β in the cell supernatant after 3 days of
cultivation of each group (n = 5 biologically independent samples). e
Representative TEM images of the cell ultrastructure in CSps, and 3
biologically independent samples were observed. N: nucleus, yellow
arrows indicate mitochondria, yellow asterisks (*) indicate endoplasmic
reticulum, and yellow triangles (△) indicate microvesicles. f
Annexin/PI analysis results of the cell survival rate (n = 3
biologically independent samples). g Proliferation assay of the CDCs
isolated from the PS-CSps, ULA-CSps, and OPC-CSps (n = 5 biologically
independent samples). h The mRNA transcription levels of Oct4, Nanog,
and Sox2 (n = 5 biologically independent samples). i The mRNA
transcription levels of VEGF, bFGF, HGF, and IGF-1 (n = 5 biologically
independent samples). All data are shown as the mean ± SD, *P < 0.05
vs. PS, ^#P < 0.05 vs. ULA, one-way ANOVA (d, f) or two-way ANOVA (b,
g, h, i).
OPC substrate improved the mitochondrial function of CSps by enhancing
oxidative phosphorylation and decreasing glycolysis
The results of RNA-sequencing (RNA-Seq) analysis showed that there were
1251 differentially expressed genes (DEGs) between the ULA group and
the OPC group (Fig. [103]4a), and 232 DEGs were not among the DEGs of
PS vs. ULA or PS vs. OPC (Fig. [104]4b). Kyoto Encyclopedia of Genes
and Genomes (KEGG) enrichment analysis showed that metabolic pathways,
glycolytic/glycogenic pathway, and the HIF-1 signaling pathway are the
top 3 significant signaling pathways of the DEGs between the OPC-CSps
and the ULA-CSps (Fig. [105]4c). Moreover, gene set enrichment analysis
(GSEA) also revealed the downregulation of the hypoxic and glycolytic
components in the OPC-CSps compared to the ULA-CSps (Fig. [106]4d). The
oxidative phosphorylation genes in the OPC group, including CS, COXII,
IDH2, SDHA, and MDH2, were notably upregulated compared with those in
the PS and ULA groups (P < 0.05). Compared to the ULA group, the key
genes of the glycolytic pathway, HK2, LDHA, and PFKL, dramatically
decreased in the OPC group (P < 0.05) (Fig. [107]4e). In addition, the
transcription levels of these three genes showed no difference between
the PS group and the OPC group. Compared to the PS-CSps and the
ULA-CSps, the 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
amino)−2-deoxyglucose (2-NBDG) uptake level of the OPC-CSps
significantly decreased (P < 0.05) (Fig. [108]4f). The lactate
production by the OPC-CSps was markedly lower than that by the
ULA-CSps, and it was significantly higher than that by the PS-CSps
(P < 0.05) (Fig. [109]4g). Among these three groups, the OPC-CSps had
the highest ATP production level (Fig. [110]4h).
Fig. 4. OPC substrate improved the mitochondrial function of CSps by
enhancing oxidative phosphorylation and decreasing glycolysis.
[111]Fig. 4
[112]Open in a new tab
a Hierarchical cluster analysis of upregulated (red) and downregulated
(blue) genes after culture on different substrates for three days. b
The DEGs from the hierarchical cluster analysis were interpreted in the
Venn diagram. The DEGs with a |log2FC| ≥ 1 and a non-adjusted P value ≤
0.05 were identified by DESeq (1.28.0). c KEGG analysis of the top 7
significant pathways (P value < 0.05). d GSEA revealed that the genes
of the hallmark MSigDB collection were mainly enriched in hypoxia- and
glycolysis-related pathways. NES, normalized enrichment score; NOM p,
nominal P value; FDR q, false discovery rate q value. e mRNA
transcription levels of the genes in the metabolic oxidative
phosphorylation pathway (CS, COXII, IDH2, SDHA, and MDH2) and the
glycolytic pathway (HK2, LDHA, and PFKL) (n = 5 biologically
independent samples). f Measurement of glucose uptake by CSps using
2-NBDG (n = 3 biologically independent samples). g Lactate release
level of CSps (n = 5 biologically independent samples). h The ATP
levels of CDCs isolated from the CSps of each group (n = 3 biologically
independent samples). i Representative TEM images of the mitochondrial
morphology from each group, and the density of the mitochondria from
each group was quantified (n = 8 images from 2 experiments). j
Rhodamine 123 staining results of mitochondrial membrane potential, and
the fluorescence intensity of the CDCs isolated from CSps of each group
were measured (n = 15 images from 3 experiments). All data are shown as
the mean ± SD, *P < 0.05 vs. PS, ^#P < 0.05 vs. ULA. One-sided
student’s t test (b), one-sided hypergeometric test with
Benjamini–Hochberg multiple testing correction (c), one-way ANOVA (f–j)
or two-way ANOVA (e).
The TEM results showed that the mitochondria in the CDCs of ULA-CSps
exhibited obvious swelling, vacuolization, and cristae breakage, while
the mitochondria in the CDCs of OPC-CSps and the PS-CSps maintained
normal cristae morphology. Furthermore, the density of mitochondria was
significantly increased in the OPC group compared to the PS group and
the ULA group (P < 0.05) (Fig. [113]4i). Mitochondrial membrane
potential levels of the CDCs in CSps were evaluated by
immunofluorescence staining of rhodamine 123 fluorescence intensity,
and the membrane potential level was significantly enhanced in the
cells of OPC group compared to those of the PS group and the ULA group
(P < 0.05) (Fig. [114]4j).
OPC-CSps showed enhanced oxidative stress resistance and anti-inflammatory
effect
H[2]O[2] stimulation was used to test cellular oxidative stress
resistance. As shown in Fig. [115]5a, b, after exposure to H[2]O[2] for
24 h, the fluorescence intensity of the ULA group was significantly
lower than that of the PS group(P < 0.05). Moreover, the fluorescence
intensity of the OPC group further decreased compared to the PS group
and the ULA group, suggesting that OPC-CSps generated the least
reactive oxygen species (ROS) and superoxide under an oxidative stress
environment. Following 12 h of H[2]O[2] stimulation, the percentage of
viable cells in the OPC group (73.7 ± 1.4%) was significantly higher
than that in the PS group (59.7 ± 7.5%) and the ULA group (59.2 ± 5.4%)
(P < 0.05) (Fig. [116]5c). There was no difference in the cell survival
rate between the PS-CSps and the ULA-CSps. Following 24 h of H[2]O[2]
stimulation, a similar trend in the survival rate was observed among
the three groups, and the survival rates of the PS, ULA, and OPC groups
were 1.5 ± 0.7%, 5.4 ± 2.1%, and 31.7 ± 4.7%, respectively
(Fig. [117]5d). The anti-inflammatory effects were also examined.
Following 12 h of TNF-α stimulation, the survival rates of the PS, ULA,
and OPC groups were 50.0 ± 3.7%, 53.8 ± 4.5%, and 66.3 ± 3.0%, which
were significantly lower than the control group, while the cells from
the OPC-CSps group exhibited the best anti-inflammatory effect among
three groups (Fig. [118]5e).
Fig. 5. Improvement in oxidative stress resistance in OPC-CSps following
H[2]O[2]-induced injury.
[119]Fig. 5
[120]Open in a new tab
Representative images and corresponding quantitative results of the (a)
ROS and (b) superoxide fluorescence intensity following 24 h of
H[2]O[2]stimulation (n = 15 images from 3 experiments). The percentages
of viable cells from each group following (c) 12 h and (d) 24 h
H[2]O[2]stimulation were determined by Annexin V/PI flow cytometry
analysis (n = 3 biologically independent samples). e The percentages of
viable cells from each group following 12 h of TNF-α stimulation were
determined by Annexin V/PI flow cytometry analysis (n = 3 biologically
independent samples). All data are shown as the mean ± SD, *P < 0.05
vs. Control, ^#P < 0.05 vs. PS, ^&P < 0.05 vs. ULA, one-way ANOVA.
Long-term cardiac function improvement following OPC-CSps transplantation in
a rat MI model
In vivo live imaging was performed to detect the survival rate of
transplanted OPC-CSps within the infarct area. The survival rate of
transplanted OPC-CSps was 53.83 ± 9.01% at week 2 and 16.18 ± 3.68% at
week 4 (Fig. [121]6a, b). According to the echocardiography results,
serious motor dysfunction of the anterior ventricular wall was observed
in the vehicle group following ligation. However, motor function was
maintained in the OPC-CSps group compared to the vehicle group. The
results also revealed that the OPC-CSps group significantly improved
cardiac function starting at week 4, and this tendency was sustained
throughout the 12-week observation (Fig. [122]6c). Compared to the
vehicle group, the OPC-CSps group showed a remarkable increase in left
ventricular fractional shortening (LVFS) and left ventricular ejection
fraction (LVEF) (P < 0.05) (Fig. [123]6d, f). The LVEF and LVFS at week
12 relative to week 4 of the vehicle group decreased by 2.74 ± 2.33%
and 2.51 ± 1.24%, respectively. In contrast, the OPC-CSps group showed
a 10.12 ± 2.57% improvement in LVEF and 4.54 ± 2.33% in LVFS at week 12
relative to week 4 (Fig. [124]6e, g). In addition, compared to the
vehicle group, the OPC-CSps group showed a significant reduction in
left ventricular internal diameters both in systole (LVIDs) and
diastole (LVIDd) at week 8 and week 12 (P < 0.05) (Fig. [125]6h–k).
Fig. 6. OPC-CSps improved long-term MI cardiac functions.
[126]Fig. 6
[127]Open in a new tab
a Representative image of DiR-labeled OPC-CSps at the instant, 14th,
and 28th day after transplantation. b Quantification results of the
survival of OPC-CSps in vivo at 28 days. (*P < 0.05 vs. D0, ^#P < 0.05
vs. D14, n = 8 rats). c Representative M-mode echocardiography images
of the sham group, the vehicle group, and the OPC-CSps group. d LVEF of
each group over 12 weeks. e The relative changes in LVEF at week 12
relative to week 4. f LVFS of each group over 12 weeks. g The relative
changes in LVFS at week 12 relative to week 4. h The change in LVIDs
over 12 weeks. i The relative changes in LVIDs at week 12 relative to
week 4. j The change in LVIDd over 12 weeks. k The relative changes in
LVIDd at week 12 relative to week 4. All data are presented as the
mean ± SD, *P < 0.05 vs. sham, ^#P < 0.05 vs. vehicle, Two-sided
student’s t test (e, g, i, k), one-way ANOVA (b) or two-way ANOVA (d,
f, h, j). For (d–k), n = 8 rats.
OPC-CSps protected infarcted myocardium from inflammation, apoptosis, and
hypertrophy
Compared to the vehicle group, the OPC-CSps group exhibited a
significant cardioprotective effect (Fig. [128]7a, b). Clear vascular
structures at the infarct region border were observed in the vehicle
group and the OPC-CSps group. However, compared to the vehicle group,
there were fewer perivascular collagens in the OPC-CSps group
(Fig. [129]7a). Compared to the sham group, an increase in the size of
the infarct area (29.25 ± 3.63%) and a decrease in myocardial tissue
retention (23.66 ± 3.52%) were observed in the vehicle group. Compared
to the vehicle group, the OPC-CSps group showed a significant decrease
in infarct area size (18.54 ± 3.19%) and an increase in retained
myocardial tissue (41.69 ± 7.99%) (Fig. [130]7c, d). Compared to the
vehicle group (0.60 ± 0.06 mm), the thickness of the left ventricular
wall was higher in the OPC-CSps group (1.27 ± 0.19 mm), which reached
46% of the normal left ventricle wall thickness (2.71 ± 0.28 mm) (Fig.
[131]7e).
Fig. 7. The reduction in cardiac inflammation, apoptosis, and hypertrophy
after OPC-CSps transplantation.
[132]Fig. 7
[133]Open in a new tab
a Representative images of Masson trichrome staining 12 weeks following
MI, and yellow arrows mark the blood vessels in the border of the
infarct area. (n = 8 rats). b Representative images of CD68^+
macrophages in the peri-infarct zone 2 weeks following MI, α-SMA^+
CD31^+ vessels in the infarct zone 12 weeks following MI, and TUNEL^+
cells in the peri-infarct zone 12 weeks following MI (n = 15 images
from 3 rats). Spilt channels of the α-SMA and CD31 staining results are
shown in Supplementary Fig. [134]4 (n = 15 images from 3 rats). c
Quantitative results of the infarct area (n = 8 rats). d The percentage
of viable myocardium at the infarct area (n = 8 rats). e Quantitative
results of infarct wall thickness (n = 8 rats). f The quantitative
results of CD68^+ macrophages per HPF assessed by ImageJ software
V1.8.0.112. HPF high-power field (n = 15 images from 3 rats). g The
quantitative results of vessel density of each group (n = 15 images
from 8 rats). h The quantitative results of the TUNEL^+ rate assessed
by ImageJ software V1.8.0.112 (n = 15 images from 8 rats). i
Representative images of WGA staining of heart tissues shown in
different regions at 12 weeks. The cardiomyocyte membrane was stained
with WGA (green), cardiomyocytes were identified by staining for cTnT
(red), and DAPI showed nuclei. Quantitative analysis of cardiomyocyte
cross-sectional area from j the left ventricle, k the border zone, and
l the remote zone (n = 15 images from 8 rats). Each data point is
represented as the mean ± SD, *P < 0.05 vs. sham, ^#P < 0.05 vs.
vehicle, Two-sided student’s t test (c, d) or one-way ANOVA (e, f, g,
h, j, k, l).
For cardiac inflammation evaluation, CD68^+ macrophages were
calculated. In the sham group, macrophage infiltration was scarcely
observed, and the number of CD68^+ macrophages in the vehicle group
significantly increased compared to that in the sham group (P < 0.05).
In the OPC-CSps group, the number of CD68^+ macrophages significantly
decreased compared to that in the vehicle group (P < 0.05)
(Fig. [135]7b, f). The structure and distribution of the vessels in the
LV wall were observed by α-SMA and CD31 immunofluorescence co-staining.
The vessel lumen could be obviously observed in the sham, vehicle, and
OPC-CSps groups. Compared to the sham group, the vessel density of the
vehicle group and the OPC-CSps group both significantly increased
(P < 0.05). Compared to the vehicle group, a significantly higher
vascular density (P < 0.05) and mature large-diameter blood vessels
(>100 μm) were observed in the OPC-CSps groups (Fig. [136]7b, g,
Supplementary Fig. [137]4). Terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) staining results
showed that the percentage of apoptotic cells in the vehicle group
(62.71 ± 10.17%) was significantly higher than that in the sham group,
and a significant decrease was observed in the OPC-CSps group (29.11 ±
9.54%) (P < 0.05) (Fig. [138]7b, h).
As the wheat germ lectin (WGA) staining results showed, the average
cardiomyocyte cross-sectional area was significantly higher in the
infarct zone, border zone, and remote zone in the vehicle group than in
the sham group (P < 0.05). However, in the border zone and the remote
zone, the OPC-CSps group exhibited smaller cardiomyocyte sizes than the
vehicle group (P < 0.05), and no significant differences were observed
between the sham group and the OPC-CSps group in all areas measured
(Fig. [139]7i–l).
Discussion
Substantial progress in MI cell therapy has been widely reported, and
improving transplanted cell survival and therapeutic outcomes remain
the key issues to be addressed. Optimizing the culture system had great
significance in obtaining abundant transplanted cells with favorable
bioactivities. This study aimed to improve the therapeutic potential of
CSps by optimizing their culture substrate. The prepared OPC substrate
was a kind of cholesteric liquid crystal obtained by the esterification
between HPC and OC. When cultured on the OPC substrate, CDCs could
spontaneously form homogenous 3D spheroids at a high density, which was
beneficial for quickly acquiring sufficient CSps in clinical
applications. Compared with the PS substrate and the ULA substrate,
CSps cultured on the OPC substrate could be activated and exhibited a
superior paracrine effect, enhanced metabolic state, and improved
oxidative stress resistance in the unique pyroptosis microenvironment.
In a rat MI model, CSps prepared by OPCs showed a long-term
cardioprotective effect within 12 weeks. A decrease in host cell
apoptosis, improvement in angiogenesis, and reduction in ventricular
remodeling were observed following OPC-CSps transplantation.
For producing highly functional CSps, preparing the proper cell culture
substrate is the first and vital step. In this study, liquid-crystal
OPC was synthesized using HPC as the rigid chain and OC as the flexible
chain (Fig. [140]1). An optical texture formed by lattice defects was
observed under the polarizing microscope, which was due to the
characterization of entropy-induced phase transitions of OPC. After
being subjected to an external shear force, the rigid chains of liquid
crystal materials maintain molecular orderliness, while the flexible
chains adjust their orientation in response to mechanical variations.
The change in orientation of flexible chains not only improved the
orderliness of materials but also drove the change in molecular
position ordering of rigid chains when such changes accumulated to a
certain extent. These alterations eventually led to the formation of
lattice defects inside the material and further resulted in texture
changes. The increase in the crystallinity index following shear force
application implied an improvement in the orderliness of the materials,
and the change in grain size was related to the change in the molecular
position of the rigid chain.
In addition, the complex phase behavior of OPC is determined by the
structural mosaic of rigid chains and flexible chains. Within the
detection range of rheological characterization, a phase transition was
observed in OPC. In the 1–10 rad region, the stress hysteresis of the
flexible chain resulted in the tendency of viscous deformation. In the
10–100 rad region, the rigid chain dominated, and the stress hysteresis
of the flexible chain attenuated, leading to a higher tendency of
elastic deformation in OPC. The OPC substrate exhibited the highest
CSps production efficiency among the three groups (Fig. [141]2c). The
density of CSps in the OPC group was 500 times greater than that in the
PS group, while it was 10 times higher than that in the ULA group.
These findings showed that OPC could rearrange the liquid crystal units
in response to external stress, and the characteristics of mechanical
responsiveness could promote CSps formation, which could satisfy the
demand for large-scale CSps production in clinical applications.
The sizes of 3D spheroids have a significant influence on cell
viabilities. Spheroids within 200 μm in diameter could grow under
sufficient nutrition and oxygen supply, which was beneficial for
delaying the formation of hypoxic cores and maintaining the viability
of their internal cell populations^[142]41,[143]42. In this study,
different sizes of CSps were observed in the PS, ULA, and OPC groups.
CSps cultured on the OPC substrate were observed to form 70–140 μm in
diameter within five days (Fig. [144]2b), and the internal cells in the
OPC-CSps maintained normal cell ultrastructure (Fig. [145]3e). In
contrast, the ULA spheroids were 100-470 μm in diameter and beyond the
size of effective nutrient and oxygen transportation^[146]43, so the
ULA-CSps showed serious cell damage in the core of ULA-CSps with a
higher portion of apoptosis (Fig. [147]3e, f). Owing to the mechanical
cues from OPC, massive CSps with controllable size and favorable
bioactivity could be obtained effectively.
In addition, cell‒cell and cell-ECM contacts also greatly affect the
phenotypes of the cells in CSps. Compared to the PS group, the
expression of CD31 and CD34 significantly increased in the ULA group
and the OPC group (Fig. [148]2d), and the focal adhesion pathway of
these 2 groups significantly differed from the PS group, which could be
related to their higher efficiency in 3D spheroid formation. It is
widely studied that the ECM components and the cell-cell contacts
within the 3D cells spheroids have significant changes than the 2D
culture, and these results showed CDCs in 3D spheroids may enhance
their phenotypes towards endothelial cells via the change of
microenvironmental cues in the ECM^[149]44, but more studies are needed
to verify this assumption. The different portions of CD90^+ cells could
be observed when cultured on the PS, ULA, and OPC substrates, so it is
reasonable to assume that the expression level of CD90 could be
regulated by the physical and chemical environments provided by each
substrate. In addition, several studies have shown that a decrease in
mesenchymal markers, such as CD90, could lead to an improvement in the
pluripotency of spheroid cells^[150]45,[151]46. In this study, compared
with the PS group and the ULA group, the OPC group showed the lowest
expression level of CD90 and the highest expression levels of
pluripotency markers, including Nanog, Sox2, and Oct4 (Fig. [152]3h).
Furthermore, the highest expression of the cardiovascular progenitor
marker KDR and the cardiac fibro-adipogenic progenitor marker Sca-1 was
observed in the OPC group (Fig. [153]2d). In conclusion, culturing CDCs
on the OPC substrate could not only obtain more progenitors in the CDC
population but also facilitate their differentiation into the
endothelial lineage.
In addition to favorable cell bioactivities, improving cellular
resistance to oxidative stress and the inflammatory environment is
another key issue in cell therapy. Following acute MI, the degradation
of the extracellular matrix and the cytokines released by dead
cardiomyocytes lead to serious inflammation, and massive host cells
induce pyroptosis^[154]47. Caspase-1 and IL-1β are the classical
factors of the pyroptosis signaling pathway. In this study, pyroptosis
of the external cells of CSps was induced by mechanical cues from OPC,
with the activation of caspase-1 and an increase in the release of
IL-1β (Fig. [155]3a–d). Meanwhile, by receiving the proper stimulation
from external pyroptosis, the internal cells with higher bioactivities
exhibited an improved paracrine effect, and improved VEGF, HGF, IGF-1,
and bFGF were observed (Fig. [156]3i). In addition, compared with the
hypoxic microenvironment in CSps from the ULA group, the cellular
proliferation activity of OPC-CSps was maintained (Fig. [157]3g).
Therefore, these results demonstrated that the OPC substrate could
provide proper stimulation for CSps to improve cellular paracrine
effects and maintain their proliferation ability.
Cellular inflammation is directly related to oxidative stress, and
improving antioxidative stress ability is vital for cell survival in MI
cell therapy. When exposed to oxidative stress, the generated hydroxyl
radicals can react with all biological macromolecules, causing DNA,
protein, membrane damage, and ultimately cell death. Mitochondria are
the center of energy metabolism, and they control many signals in cell
fate programs^[158]48. It was reported that enhancing mitochondrial
respiration and function could reduce the damage caused by oxidative
stress^[159]49. In this study, compared to the PS group and the ULA
group, the OPC-CSps exhibited higher mitochondrial density and membrane
potential levels (Fig. [160]4i, j). It was reported that cells in
hypoxia would lead to mitochondrial damage^[161]50, and with a compact
core in the CSps, the ULA group showed lower oxidative stress
resistance. In contrast, under the proper stimulation induced by OPC,
the CSps in the OPC group could acquire the ability to resist oxidative
stress before transplantation (Fig. [162]5). Taking these results
together, CSps with improved oxidative stress resistance could be
obtained using OPC as the culture substrate.
Furthermore, RNA-seq analysis was employed to investigate the
underlying mechanism of differences in cell bioactivity and oxidative
stress resistance among the three groups (Fig. [163]4a). The metabolic
level and bioenergetic state are highly related to the availability of
oxygen and nutrients^[164]51. In this study, DEGs between the OPC group
and the ULA group were significantly enriched in the
glycolysis/gluconeogenesis pathway and HIF-1 signaling pathway, but
these pathways were not in the top 7 enriched pathways between the OPC
group and the PS group (Fig. [165]4c, d). These results further proved
that the structure of the OPC-CSps could satisfy the demand for
internal CDC metabolism. It was reported that the enhancement of
oxidative phosphorylation could surmount mitochondrial fission and
functional failure^[166]52, while glycolysis was associated with
mitochondrial dysfunction^[167]53. In this study, the oxidative
phosphorylation of OPC-CSps was enhanced, while the ULA-CSps altered
their energy production toward glycolysis (Fig. [168]4e). Therefore,
the highest ATP level was observed in the OPC group (Fig. [169]4h), and
the OPC-CSps showed improved mitochondrial function and effective
protection against cell damage in oxidative stress. In addition, the
OPC group showed a significant decrease in glucose uptake levels
(Fig. [170]4f), suggesting that the OPC-CSps may survive longer in
nutrient-limited conditions. In conclusion, these results illustrated
that CSps could switch toward a highly metabolically active state when
cultured on the OPC substrate, which is beneficial for OPC-CSps’
long-term survival in the hostile microenvironment.
With favorable cell viabilities, superior antioxidative stress, and
long-term cellular survival ability, the therapeutic effect of OPC-CSps
on MI was evaluated. As the results showed, the OPC-CSps group
significantly improved MI cardiac function. Compared with the overtime
decline tendency of the vehicle group, a consistent increase in left
ventricular systolic and diastolic function was observed in the
OPC-CSps group during the 12-week observation (Fig. [171]6). To pursue
favorable outcomes in MI therapy, reducing myocardial inflammation and
rebuilding the vessel network are crucial issues for protecting cardiac
structure and function. In this study, owing to the effective
preconditioning treatment in vitro, the OPC-CSps acquired enhanced
inflammation resistance and improved regenerative function. The
transplanted CSps were tracked by Dir labeling, and the results showed
that 16.18% ± 3.68% of CSps survived 4 weeks following transplantation
(Fig. [172]6a, b). Meanwhile, a significant decrease in CD68^+
macrophages in the border zone was observed in the OPC-CSps group
(Fig. [173]7b, f). Moreover, effective angiogenesis within the
infarcted myocardium was widely observed at 12 weeks following MI
(Fig. [174]7b, g). Therefore, these in vivo results demonstrated that
transplanting OPC-CSps could achieve satisfactory long-term cardiac
function recovery by effectively reducing myocardial inflammation and
promoting angiogenesis.
Furthermore, protecting normal cardiac structure was a key issue for
maintaining MI cardiac function. Owing to the severe inflammation and
the hostile environment following MI, excessive degradation or impaired
synthesis of ECM after MI was considered to accelerate ventricular
remodeling, including myocardial fibrosis and cardiac hypertrophy, and
ultimately lead to heart failure^[175]54. Cardiac fibrosis greatly
reduces cardiac function by replacing necrotic myocardial tissue with
enlarged scars. In addition, massive cell death, a decrease in
contractile activity in the affected zone, and increased hemodynamic
burden were assumed to be the main causes of cardiac
hypertrophy^[176]55. In this study, transplantation of OPC-CSps
significantly decreased the infarct area, and an increase in viable
cardiac tissue was observed (Fig. [177]7a, c, d), showing a beneficial
effect in preventing cardiac hypertrophy (Fig. [178]7i–l). These
results supported that transplanting OPC-CSps could greatly protect MI
cardiac function by reducing ventricular remodeling.
In summary, to improve the MI therapeutic potential of CSps, a novel
cell culture substrate, liquid crystal OPC, was prepared for CSps
culture and preconditioning. The OPC substrate served as a special
mechanical cue to effectively promote the formation of CSps of
controllable size. Furthermore, the OPC-CSps exhibited significant
enhancement of biological function and antioxidative stress abilities.
In the rat MI model, OPC-CSps not only showed great cell retention and
survival in the infarct area but also significantly improved cardiac
wall thickness, angiogenesis, and long-term cardiac function. In
conclusion, using the OPC substrate could satisfy the demand for
large-scale CSps production with excellent cardiac regeneration
abilities for MI therapy.
Methods
Animals
Rats were housed in specific pathogen-free conditions with 12 h
day/light cycles. Rats were healthy and had free access to water and
food. All animal studies were performed in accordance with the ethical
guidelines of the National Guide for the Care and Use of Laboratory
Animals and approved by Jinan University Animal Care and Use Committee
(Approval numbers: IACUC-20210113-06). Four-week-old male Sprague
Dawley rats (Guangdong Medical Laboratory Animal Center) were used for
isolating primary CDCs, and 3-month-old female Sprague Dawley (Vital
River) rats were used to establish myocardial infarction animal models.
Every attempt was made to minimize the use of animals and pain.
Preparation of the octyl hydroxypropyl cellulose ester (OPC) substrates
OPC was prepared via esterification between hydroxypropyl cellulose
(HPC) (Sigma‒Aldrich, Mw = 100,000 g/mol) and octanoyl chloride (OC)
(Sigma‒Aldrich). Briefly, 5.0 g HPC was dissolved in 30 mL dehydrated
acetone with mild stirring. Seven milliliters OC was added to the
solution when HPC dissolved completely, and the reaction was kept at
55 °C for 4 h. Then, 300 ml distilled water was added to the reaction
mixture, and a cream color sticky mass was obtained after removing the
liquid phase. The cream-colored sticky mass was dissolved in acetone
and precipitated by adding water to the solution. This step was
repeated 6 times. After dissolving in ethanol and dialyzing in
distilled water 15 times to remove the residual OC, OPC could be
obtained after precipitation. Finally, the OPC product was dried in a
vacuum at 55 °C for 48 h.
For the preparation of the OPC substrate, a 3% OPC concentration
mixture was obtained by stirring OPC and ethanol for 1 h at 20 °C. It
was cast onto clean culture dishes. After the solvents evaporated at
room temperature, the dishes were washed with distilled water 10 times
for 4 h each time. Then, the dishes were sterilized by Co60 irradiation
(15kGy).
Characterization of OPC
The surface characteristics of OPC were observed by polarized optical
microscope (Carl Zeiss Axioskop40). An atomic force microscope
(BENYUAN) was used to analyze the surface roughness in on-contact mode.
The measurement of the water contact angle on the OPC substrate was
tested at room temperature by a contact angle meter (Kruss DSA100) with
ultrapure water as the testing liquid and a humidity of 80%.
The changes in the OPC structure when subjected to a shear force were
tested by X-ray diffraction (XRD, Dmax1200). Briefly, 3% OPC solution
was added to the glass surface and then covered with the magnesium
sheet. Next, the samples were dried by placing them in a vacuum at
55 °C for 48 h. Then, the magnesium sheet was slid by weights of equal
mass with a distance of 3 mm. The XRD patterns were recorded from 5° to
40° at a step width of 0.02° and scanning speed of 8°/min.
The crystallinity index (Cr. I) of OPC was determined according to the
Segal method and calculated using Eq. ([179]1)^[180]56
[MATH:
Cr.I=I002−Iamorph
I002 :MATH]
1
where I[002] is the maximum intensity of the main diffraction, and
I[amorph] is the intensity of the amorphous background scatter measured
at 2θ = 18° where the intensity is minimum.
The crystallite diameter (D[002]) perpendicular to the (002) plane was
calculated from the Scherrer Eq. ([181]2)^[182]57
[MATH:
D002=
kλβcosθ :MATH]
2
where K is a Scherrer constant that equals 0.9, λ is the wavelength of
the radiation (1.54 Å for CuKα), β is the width of the peak at half
maximum, and θ is the angle of incidence.
Finally, the rheological properties of OPC were measured by a DHR-2
stress-controlled rheometer (TA Instruments). The oscillation-frequency
mode at 37 °C was incorporated for rheological tests with a strain of
1% and ω = 1–100 rad s^−1.
For the OPC toxicology test, 2 × 10^3 CDCs were seeded in 96-well
plates and cultured in 100 μl of CSps culture medium or OPC immersion
culture medium. Ten microliters of CCK8 (Dojindo) was added to each
well and incubated for 2 h every 24 h for five consecutive days, and
the optical density values were recorded at 450 nm wavelengths.
Isolation and culture of CDCs
Cardiac tissue specimens from the septum of the left ventricle were
minced and digested with collagenase IV (Sigma) for 20 min at 37 °C.
These tissues were plated on poly-d-lysine (Sigma)-coated dishes in
CSps culture medium, which consisted of 500 ml Iscove DMEM (Corning),
1% l-glutamine (Corning), 1% penicillin‒streptomycin solution
(Corning), 10% fetal bovine serum (BD Bioscience) and 0.1 mmol/L
β-mercaptoethanol (Gibco). After 1–2 weeks, a monolayer of adherent
cells that grew out from these tissues was harvested by 0.05% trypsin
and passaged on poly-d-lysine-coated dishes. Following 3–7 days of
cultivation, the CSps were collected and plated onto fibronectin-coated
dishes and expanded as monolayer CDCs. All cultures were cultured in 5%
CO[2] at 37 °C.
Preparation of CSps
Polystyrene (PS) substrate, ultralow attachment (ULA) substrate, and
OPC substrate were used to culture CDCs and obtain CSps. Cells were
seeded onto three different substrates at a density of
7 × 10^4 cells/cm^2. The formation of CSps in each group was observed
by an inverted microscope (Olympus IX71) for 5 consecutive days. The
diameter and the total number of CSps at the same time point were
measured using ImageJ software V1.8.0.112, and at least 30 CSps from
each group were randomly chosen.
Flow cytometry test
The expression levels of the CSps surface markers CD31 (1:200,
GB11063-3, Servicebio), CD34 (1:200, ab81289, Abcam), CD90 (1:200,
ab225, Abcam), CD105 (1:200, ab156756, Abcam), Sca-1 (1:200, ab51317,
Abcam), and KDR (1:200, sc6251, Santa Cruz) were determined by flow
cytometry. After a 3-day cultivation, cells and CSps from different
substrates were obtained and digested into single cells. After
incubation with the primary antibodies for 1 h and the corresponding
secondary antibodies for 30 min, Alexa Fluor 488 goat anti-mouse IgG
(1:200, ab150113, Abcam) or Alexa Fluor 488 goat anti-rabbit IgG
(1:200, ab150077, Abcam) was used. The staining results were analyzed
by flow cytometry (BD FACSCanto), and a negative isotypic control was
used during the analysis.
Ultrastructure analysis
The cells and CSps from each substrate were harvested and fixed with
2.5% glutaraldehyde overnight at 4 °C. Following dehydration with a
series of graded ethanol solutions, samples were immersed in 1% osmium
tetroxide for 2 h. Next, the samples were embedded in resin and cut to
60 nm thickness. Then, the cell ultrastructure was visualized and
photographed with transmission electron microscope (TEM, JEOL).
Cell proliferation, apoptosis, and enzyme-linked immunosorbent assay (ELISA)
Cell proliferation was determined by CCK8 (Dojindo) after being
cultivated for 3 days on each substrate. Cell apoptosis rates were
determined by using the Alexa Fluor® 488 Annexin-V/Dead Cell Apoptosis
Kit (Elabsicence) according to the manufacturer’s instructions. The
results were analyzed and recorded by flow cytometry (BD FACSCanto).
Survival cells in the Q4 gate were quantified and analyzed. Following 3
days of cultivation, the culture supernatants from each group were
harvested, and the IL-1β concentration was measured by rat IL-1β ELISA
kits (MEIMIAN).
Real-time quantitative PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent. High-Capacity cDNA
Reverse Transcription Kits (Thermo Fisher Scientific) were used for
cDNA synthesis according to the manufacturer’s instructions. qRT-PCRs
were performed on a Mini Cycler PCR instrument with SYBR Green reagent
(Toyobo). Gene expression was normalized to GAPDH mRNA, and relative
expression was calculated by 2^−ΔΔCT. The primer sequences of the
detected genes are listed in Supplementary Table [183]1, and the
primers were synthesized by Guangzhou Generay Biotechnology Company.
Western blot analysis
The cells and CSps cultured on the PS, ULA, and OPC substrates were
lysed in RIPA buffer (Solarbio) for protein extraction. The protein
concentrations of the samples were adjusted by bicinchoninic acid (BCA)
protein assay (Thermo Fisher Scientific) according to the
manufacturer’s protocol. Forty micrograms of protein was loaded into
each lane of a 10% sodium dodecyl sulfate‒polyacrylamide gel
electrophoresis (Millipore) gel and transferred to polyvinylidene
fluoride membranes (Millipore). After being blocked with 5% bovine
serum albumin in Tris buffered saline Tween (Biosharp) at room
temperature for 1 h, the membranes were incubated with the primary
monoclonal antibodies against caspase-1 (1:500, ab1872, Abcam) and
β-actin (1:1000, ab8226, Abcam) in TBST overnight at 4 °C, after which
HRP-labeled Anti-Rabbit IgG antibody (1:2000, 7074, Cell Signaling
Technology) and HRP-labeled Anti-mouse IgG antibody (1:2000, 7076, Cell
Signaling Technology) was added and incubated for another 1 h. The
results were visualized via enzyme-linked chemiluminescence by an ELC
kit (Thermo Fisher Scientific). β-Actin was used as an internal
control.
Metabolic analysis of CSps
The glucose uptake ability of CDCs was evaluated by using the
fluorescent glucose 2-NBDG (Cayman). After 3 days of cultivation of
CDCs on different substrates, all the culture medium was removed and
replaced with glucose-free DMEM containing 50 µM 2-NBDG for 30 min.
Consequently, the fluorescence intensity of the cells was measured by
flow cytometry (BD FACSCanto). Following a 3-day cultivation, the
culture medium of each group was collected to test the extracellular
lactate contents, and the reagent kit was the Lactate Colorimetric
Assay Kit (Nanjing JianCheng).
For the intracellular ATP content assay, CDCs were cultured on
different substrates for 3 days and formed CSps, and single cells were
isolated from the CSps of each group and seeded in a 96-well plate at a
density of 1 × 10^5. The intracellular ATP content was determined by
using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). For
the mitochondrial membrane potential measurement, digested cells were
washed twice with PBS and incubated with 2 µM Rho123 (Beyotime) for
20 min in a dark environment at 37 °C. Fluorescence images were taken
under a fluorescence microscope (Olympus, FV3000: Olympus FV31S-SW
software displayed), and the fluorescence intensity of the cells was
analyzed with ImageJ software V1.8.0.112.
RNA-Seq analysis
According to the manufacturer’s protocol, a total RNA Kit I (Omega) was
used to extract sample RNA. Sequencing was performed on the Illumina
platform. The raw RNA-seq reads were aligned to the Rattus norvegicus
genome (rn6) by hisat2 (version:2.1.0). Mapped reads were counted by
featureCounts (v.1.6.2), and gene expression was calculated by R and
the DESeq2 package. Significant differentially expressed genes (DEGs)
among cells cultured on the PS, ULA, and OPC substrates were evaluated
using DESeq (1.28.0), and genes with a |log2FC| ≥ 1 and an adjusted P
value ≤ 0.05 were selected for further analysis. Hierarchical
clustering was performed for DEGs using a heatmap. Kobas (3.0) was used
for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis. Additionally, gene set enrichment analysis (GSEA) was carried
out using GSEA v.4.2.3.
Oxidative stress resistance level measurement
Following a 3-day cultivation, CDCs formed CSps on different culture
substrate, and the CSps were subjected to H[2]O[2] stimulation for
24 h. And then singles cells were isolated from the CSps and used for
intracellular ROS and superoxide production measurement with the
cellular ROS/superoxide detection assay kit (Abcam). In short, the
isolated cells were incubated for ROS/superoxide detection for 30 min
at 37 °C. The results were observed and recorded by a laser confocal
scanning microscope (Olympus, FV3000: Olympus FV31S-SW software
displayed). In addition, after the CSps were exposed following 12 h or
24 h of exposure to H[2]O[2], CSps were digested into single cells and
used to measure the cell survival rate with the Alexa Fluor® 488
Annexin-V/Dead Cell Apoptosis Kit (Invitrogen). The results were
analyzed by flow cytometry (BD FACSCanto).
Anti-inflammation ability measurement
Following a 3-day cultivation, CDCs formed CSps on different culture
substrate, and the CSps were subjected to 50 ng/ml TNF-α stimulation
for 12 h. And then singles cells were isolated from the CSps and used
to measure the cell survival rate with the Alexa Fluor® 488
Annexin-V/Dead Cell Apoptosis Kit (Invitrogen). The results were
analyzed by flow cytometry (BD FACSCanto).
Rat MI model and CSps transplantation
The establishment of the MI model and transplantation methods were
carried out. In brief, female rats aged 3 months (220 ± 20 g) were
anesthetized with 3% isoflurane and ventilated through endotracheal
intubation. Mechanical ventilation was provided with room air at 60 to
70 breaths min^–1 using a rodent respirator (Taimeng Company).
Subcutaneous stainless-steel electrodes were used to record the
standard electrocardiogram. After shaving the chest, a left thoracotomy
was performed to expose the heart at the fifth intercostal space. The
left anterior descending coronary artery (LAD) was ligated using a 6-0
silk suture, and ischemia was confirmed by observing ST segment (or J
point) elevation and the occurrence of cardiac cyanosis. After
stabilizing for 15 min, rats in the vehicle group were transplanted
with blank Matrigel, and rats in the OPC-CSps group were transplanted
with OPC-CSps Matrigel suspensions (0.1 mL). OPC-CSps were transplanted
into the OPC-CSps group with a total number of 5 × 10^6 cells. For the
sham group, rats were subjected to the same procedure without ligation
and Matrigel injection. Finally, the animals were closed at the chest
and monitored, and antibiotics and 0.9% normal saline solution were
administered. Three rats from each group were killed at week 2 and
cardiac samples were collected to assess macrophage infiltration.
Continuous cardiac cycles were collected for 8 rats from each group.
Ex vivo imaging analysis
Prior to transplantation, CSps were labeled with 3.5 μg/ml
1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR,
Invitrogen) following the manufacturer’s instructions. The survival
status and localization of Dir-labeled CSps were observed using a
Bruker In Vivo Xtreme II Imager (Bruker). The Bruker Molecular Imaging
Software (IB5438150 Rev. B 12/12) and ImageJ software V1.8.0.112 were
applied for imaging processing and data analysis.
Echocardiogram
Cardiac function and the movement of the left ventricular wall were
measured using a Vevo 2100 ultrasonic system before surgery and at 4,
8, and 12 weeks postsurgery. After the rats were anesthetized with
isoflurane, the four-chamber and two-chamber sections of the left
ventricle were obtained by ultrasound, left ventricular end-diastolic
volume (LVEDV) and end-systolic volume (LVESV) were measured using the
trackball at the 4-chamber and 2-chamber sections of the left
ventricle, and the left ventricle ejection factor (LVEF) was calculated
by the equation: LEVF= (LVEDV-LVESV)/LVEDV*100%.
Histology and immunochemistry assay
For cell sample preparation, CSps from each group were harvested, fixed
in a 75% ethanol solution, and embedded in OCT compound, and 5μm
sections were used. For tissue sample preparation, after rats were
euthanized, the heart tissues were harvested, fixed in 4%
paraformaldehyde, embedded in paraffin, and serially sectioned at 5μm
thickness. Masson trichrome staining was performed on tissue sections.
For immunofluorescence staining, the sections were blocked with 5% goat
serum and then incubated with primary antibodies, including
anti-caspase-1(1:100, ab1872, Abcam), anti-CD68 (1:200, 360018,
Zhengneng), anti-cardiac troponin T (cTnT, 1:500, ab209813, Abcam),
anti-smooth muscle alpha-actin (α-SMA, 1:300, 41550, SAB), and
anti-CD31 (1:300, GB11063-3, Servicebio) at 4 °C overnight. After
washing with PBS 3 times, 488 nm goat anti-rabbit (1:400, ab150077,
Abcam), 594 nm goat anti-rabbit (1:400, ab150080, Abcam), 488 nm goat
anti-Mouse(1:400, ab150113, Abcam), or 594 nm goat anti-Mouse (1:400,
ab150116, Abcam) secondary antibodies were added and incubated for 2 h
at room temperature. The dilutions were 1:200 for the primary antibody
and 1:400 for the secondary antibody. Apoptosis and the cross-sectional
area of myocardial cells were assessed by terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) (Promega) and
FITC-labeled wheat germ lectin (WGA) (1:500, Thermo Fisher Scientific)
staining, respectively. DAPI showed the nuclei. All stained images were
observed and photographed by laser confocal scanning microscope
(Olympus, FV3000: Olympus FV31S-SW software displayed).
Statistical analysis
All analyses were performed using GraphPad Prism 9.0 (GraphPad Software
Inc). For comparisons of two groups, a two-sided Student’s t test was
used. Comparisons of multiple groups were made using one- or two-way
ANOVA. The KEGG pathway of genes were analyzed by one-side
hypergeometric analysis with Benjamini–Hochberg multiple testing
correction. All quantitative results were expressed as the
mean ± standard deviation, and differences with a P value < 0.05 were
considered statistically significant.
Reporting summary
Further information on research design is available in the [184]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[185]Supplementary Information^ (1.7MB, pdf)
[186]Peer Review File^ (7.7MB, pdf)
[187]Reporting Summary^ (5.5MB, pdf)
Source data
[188]Source data^ (179.3KB, xlsx)
Acknowledgements