Differentially expressed gene analysis of KF and KK
Compared with NF, KF had 668 differentially expressed genes, which are shown in a volcano plot and a pie chart (Fig. 1a, c), including 235 up-regulated genes and 433 down-regulated genes. Compared with NK, KK had 445 differentially expressed genes, which are also shown in a volcano plot and a pie chart (Fig. 1b, d), including 232 up-regulated genes and 213 down-regulated genes. Up and down regulated genes differed in their quantitative composition in KF and KK. The results showed that there were few common differentially expressed genes between KF and KK, and the unique differentially expressed genes accounted for most of the total differentially expressed genes, 89.59% and 93.07%, respectively. Given this, different regulatory genes functioned in the different transcription processes of KF and KK, indicating that KF and KK were quite different in abnormal transcription (Fig. 1e).
Functional enrichment analysis and network-based enrichment analysis of differentially expressed genes
In order to investigate the functions of differentially expressed genes, we performed gene set enrichment analysis of up- and down-regulated differentially expressed genes in KF and KK with the Metascape method [31] respectively. The analysis results were arranged in ascending order of p-value. Significant enrichment results (top 20 terms) as shown in Fig. 2, were constructed into a network according to their correlation and similarity, and then clustered and classified according to their functional correlation. In KF, up-regulated genes were significantly enriched in the following pathways or biological processes: PID AP1 PATHWAY [32], blood vessel development, IL-17 signaling pathway [33], multi-multicellular organism process, myeloid leukocyte migration, MAPK cascade [34], muscle structure development, extracellular matrix organization, epithelia cell differentiation, positive regulation of protein phosphorylation, regulation of animal organ morphogenesis, epithelial cell proliferation, negative regulation of cell population proliferation. The KF down-regulated genes were significantly enriched in the following pathways or biological processes: regulation of cytokine production [35], NABA CORE MATRISOME [36], Adaptive Immune System, Iymphocyte activation, immune effector process, regulation of small GTPase mediated signal transduction, interleukin-6 production, tissue morphogenesis, regulation of cell adhesion, Cell adhesion molecules, leukocyte migration, leukocyte activation involved in immune response. In summary, in KF tissues, biological processes such as immune system regulation, active cell proliferation, vascular development, muscle development, and responses to external stimuli and hormone signaling play an important role (Additional file 1: Fig. S2).
In KK, up-regulated genes were significantly enriched in the following pathways or biological processes: Signaling by NOTCH1 HD Domain Mutants in Cancer, cell growth, regulation of epithelial cell proliferation involved in prostate gland development, PID INTEGRIN4 PATHWAY, developmental cell growth, response to wounding, fibroblast growth factor receptor signaling pathway, vesicle transport along microtubule, Hemostasis, SARS-Cov-2 innate immunity evasion and cell-specific immune response, memory, regulation of protein transport. Among them, the Notch signaling pathway was a highly conserved intercellular signaling pathway during animal evolution, playing an important role in maintaining stem cells undifferentiated and regulating their proliferation, differentiation, and apoptosis. In the Sprague Dawley rat proliferative scar model, inhibition of Notch signaling pathway could reduce traumatic scar proliferation [37]. This was consistent with our enrichment results of up-regulated genes in KK. Therefore, inhibiting Notch signaling is likely to inhibit keloids hyperplasia by regulating the differentiation and proliferation of epidermal cells. The KK down-regulated genes were significantly enriched in the following biological processes: multicellular organismal homeostasis, muscle structure development, skin development, cellular response to inorganic substance, response to drug, cellular response to hormone stimulus, response to growth factor, interleukin-4 and Interleukin-13 signaling, negative regulation of cell differentiation, cellular response to organic cyclic compound and regulation of secretion. In summary, during the KK formation, biological processes such as Notch signaling, the muscle, skin, cell growth development and proliferation, response to hormone stimulation and maintenance of multicellular organismal homeostasis play an important role.
Construction PPI network and identification of hub genes
In KF and KK, we constructed a differentially expressed gene protein-protein interaction network based on the protein-protein interactions in the Pathway Common PPI database, shown in the Cystoscape software (Fig. 3a, b). We calculated the hub genes of the network with 10 parameterized algorithms in cytoHubba, a plug-in of Cystoscape. It can be seen that these differentially expressed hub genes interacted with a variety of proteins. DMNC, MNC, Degree, EPC, BottleNeck, EcCentricity, Closeness, Radiality, Betweenness, Stress and Clustering Coefficient values of the hub genes in KF and KK are presented in Tables (Additional file 1: Table S1 and S2). The functions of the hub genes are described in Table 1. In KF, the hub genes identified included the up-regulated genes NOG, IL6 and JUN, and the down-regulated genes POU2F1, LEF1, SYK, PIK3R1, VAMP2, NEFL and SLC24A3. Among the up-regulated hub genes, interleukin 6 (IL-6) acts as an essential factor in bone homeostasis and on vessels directly or indirectly by induction of VEGF, resulting in increased angiogenesis activity and vascular permeability, meanwhile it also has the function of regulating immunity and stem cells [38]. JUN was a key transcription factor that recognizes and binds to the enhancer heptamer motif, involved in the PID AP1 PATHWAY, may play key roles in inflammation and complications [32]. NOG is the Inhibitor of bone morphogenetic proteins (BMP) signaling. Among the down-regulated hub genes, POU2F1 was a transcription factor can activate the promoters of immunoglobulins. PIK3R1 can binds to activated (phosphorylated) protein-Tyr kinases, through its SH2 domain, and acts as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane. SYK, which is one of non-receptor tyrosine kinases, mediates signal transduction downstream of a variety of transmembrane receptors. Other genes had the function of regulating transcription factors and transporting ions or vesicles (Additional file 1: Fig. S3). In summary, the hub genes in KF act as key transcription factors or target genes, and are involved in the formation of KF by directly participating in or regulating the mediation of immune regulation, angiogenesis, and morphogenesis, or by signaling and mediating kinase participation in biological process.
In KK, the hub genes identified included the up-regulated genes APP, IL1A and NOTCH1, and the down-regulated genes PTGS2, LEP, FOS, AKR1C3, JUN, IRF1 and PRKACA. Among the up-regulated genes, IL1A, produced by activated macrophages, IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. NOTCH1, Functions as a receptor for membrane-bound ligands to regulate cell-fate determination. APP, Functions as a cell surface receptor and performs physiological functions on the surface of neurons relevant to neurite growth, neuronal adhesion, and axon genesis. Among the down-regulated genes, IRF1, which is a transcriptional regulator, displays a remarkable functional diversity in the regulation of cellular responses. LEP is a key player in the regulation of energy balance and body weight control, has central and peripheral effects was found in many tissues. FOS, Nuclear phosphoprotein which forms a complex with the JUN/AP-1 transcription factor, involved in the PID AP1 PATHWAY, may play key roles in inflammation and complications [32]. PTGS2 is a Dual cyclooxygenase and peroxidase, and plays a particular role in the inflammatory response. In summary, the hub genes in KK act as key genes or transcription factors, and are involved in the formation of KK by directly participating in or regulating cell proliferation and immune response, as well as by participating in inflammatory response and energy balance maintenance.
Table 1
Hub genes and their functions in KF and KK
Gene symbol | Description | Function |
KF hub genes |
NOG | Noggin | Inhibitor of bone morphogenetic proteins (BMP) signaling which is required for growth and patterning of the neural tube and somite. |
LEF1 | Lymphoid Enhancer Binding Factor 1 | Transcription factor that binds DNA in a sequence-specific manner |
JUN | Jun activation domain binding protein | Transcription factor that recognizes and binds to the enhancer heptamer motif 5'-TGA[CG]TCA-3' |
POU2F1 | POU domain, class 2, transcription factor 1 | Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3') and activates the promoters of the genes for some small nuclear RNAs (snRNA) and of genes such as those for histone H2B and immunoglobulins |
PIK3R1 | Phosphoinositide-3-Kinase Regulatory Subunit 1 | Binds to activated (phosphorylated) protein-Tyr kinases, through its SH2 domain, and acts as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane. |
VAMP2 | vesicle-associated membrane protein 2 | Involved in the targeting and/or fusion of transport vesicles to their target membrane |
SYK | Spleen Associated Tyrosine Kinase | Non-receptor tyrosine kinase which mediates signal transduction downstream of a variety of transmembrane receptors including classical immunoreceptors like the B-cell receptor |
IL6 | interleukin-6 | Acts as an essential factor in bone homeostasis and on vessels directly or indirectly by induction of VEGF, resulting in increased angiogenesis activity and vascular permeability |
SLC24A3 | Solute Carrier Family 24 Member 3 | Transports 1 Ca(2+) and 1 K(+) in exchange for 4 Na(+). |
NEFL | Neurofilament Light Chain | Neurofilaments usually contain three intermediate filament proteins: NEFL, NEFM, and NEFH which are involved in the maintenance of neuronal caliber |
KK hub genes |
AKR1C3 | Aldo-Keto Reductase Family 1 Member C3 | Cytosolic aldo-keto reductase that catalyzes the NADH and NADPH-dependent reduction of ketosteroids to hydroxysteroids. |
APP | Amyloid Beta Precursor Protein | Functions as a cell surface receptor and performs physiological functions on the surface of neurons relevant to neurite growth, neuronal adhesion and axonogenesis |
FOS | Fos Proto-Oncogene, AP-1 Transcription Factor Subunit | Nuclear phosphoprotein which forms a tight but non-covalently linked complex with the JUN/AP-1 transcription factor. |
IL1A | Interleukin 1 Alpha | Produced by activated macrophages, IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. |
IRF1 | Interferon Regulatory Factor 1 | Transcriptional regulator which displays a remarkable functional diversity in the regulation of cellular responses |
LEP | Leptin | Key player in the regulation of energy balance and body weight control. Once released into the circulation, has central and peripheral effects by binding LEPR, found in many tissues, which results in the activation of several major signaling pathways |
NOTCH1 | Notch Receptor 1 | Functions as a receptor for membrane-bound ligands Jagged-1 (JAG1), Jagged-2 (JAG2) and Delta-1 (DLL1) to regulate cell-fate determination. |
PRKACA | Protein Kinase CAMP-Activated Catalytic Subunit Alpha | Phosphorylates a large number of substrates in the cytoplasm and the nucleus |
PTGS2 | Prostaglandin-Endoperoxide Synthase 2 | Dual cyclooxygenase and peroxidase in the biosynthesis pathway of prostanoids, a class of C20 oxylipins mainly derived from arachidonate, with a particular role in the inflammatory response |
Enrichment function analysis on differentially expressed hub genes in KF and KK
Pathway and GO term enrichment analyses were performed for differentially expressed hub genes, and an interaction network was constructed for significantly enriched pathways and GO terms to reveal the keloid-promoting molecular mechanism centered on differentially expressed hub genes. KF hub gene mainly enrichment pathways including Signaling by interleukins, neutrophil mediated immunity, TGF-beta receptor signaling and apoptotic signaling pathway (Additional file 1: Fig. S3a, b), the KK hub gene mainly enrichment pathways including PID TCR CALCIUM PATHWAY, cellular response to external stimulus, response to corticosteroid, positive of regulation cytokine production, multicellular organismal homeostasis, negative regulation of cell population proliferation, reproductive structure development (Additional file 1: Fig. S3c, d). Similar to the enrichment results of differentially expressed genes, in the formation of KF, hub genes were significantly enriched in the following pathways: immune regulation-related biological process and signaling regulation process; in the formation of KK, hub genes were significantly enriched in the following biological processes: cell regulation, structural development and response to stimuli.
Upstream transcription factors of differentially expressed hub genes
We obtained all possible transcription factors that up-regulated hub genes from ChIP-seq experimental data of human samples in the ENCODE Project. Then, we identified hub gene transcription factors that functioned in keloids by calculating the expression correlation between these transcription factors and hub genes in our study samples. The results showed that transcription factors and hub genes presented a high gene expression correlation (Fig. 4a-f). Thus, we infer that transcription factors play a relatively important role in the formation of both KF and KK by up-regulating hub genes. Among the transcription factors regulating the expression of up-regulated genes in KF, the MYC gene, a regulatory transcription factor of NOG, is a proto-oncogene and encodes a nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis, and cellular transformation. NOG have pleiotropic effects, may play a major role in morphogenesis and development. We infer that MYC accelerates morphogenesis and development by up-regulating NOG expression. TFAP2C is the regulatory transcription factors of IL6, proteins encoded by this gene involved in the activation of several developmental genes. It plays a role in the development of the eyes, face, body wall, limbs, and neural tube, may regulate the ability of interleukin 6 (IL-6) to exert its immunoregulation and increase its angiogenic activity and vascular permeability. ATF1, a transcription factor of JUN, encodes an activating transcription factor. It influences cellular physiologic processes by regulating the expression of downstream target genes, which are related to growth, survival, and other cellular activities and enhances cell transformation. We infer that during KF formation, these transcription factors play an important role by regulating cell processes such as immune activity, angiogenesis, cell growth and cell development. Among the transcription factors regulating the expression of up-regulated hub genes in KK, ZKSCAN1, a transcription factor of APP, elevated expression of this gene has been observed in gastric cancer and the encoded protein may stimulate migration and invasion of human gastric cancer cells, may promote the migration of keloid-forming cells in KK. ELF1, a transcription factor of NOTCH1, the encoded protein is primarily expressed in lymphoid cells and acts as both an enhancer and a repressor to regulate transcription of various genes. We infer cell fate determination can be regulated by regulating NOTCH1 expression. MYC, a transcription factor of IL1A, is a proto-oncogene and encodes a nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation. It can regulate the high expression of IL1A and stimulate thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. We found that MYC was highly expressed in both KK and KF and was a common transcription factor regulating the expression of up-regulated hub genes during KK and KF formation. Therefore, we guess it is an important gene, and infer that it have the functions of regulating cell cycle, apoptosis and promoting cell growth and development in KF and KK.
Cluster analysis of hADSCs
The hADSCs samples named A20_13, A21_05 and A21_06 were obtained from three healthy adult donors, and 5121, 4652 and 4449 cells were obtained respectively after the removal of low-quality cells by filtration. In order to systematically analyze hADSCs, we integrated the three samples and removed batch effects to obtain 14,222 cells in total, and then performed normalization, dimensionality reduction, and clustering using the Seurat package. The results showed that the hADSCs could be divided into 10 clusters with different functions (Fig. 5a). The results of the clusters clustering showed significant consistency among the three replicates, demonstrating the reliability of the clustering method (Additional file 1: Fig. S5).
Identification of hADSCs clusters with therapeutic potential for inhibiting keloid formation.
We performed marker gene identification for 10 clusters, with the screening criteria for the top 200 marker genes in clusters being P < 0.05 and log2fc > 0, and we obtained marker genes in each cluster. The GPCR is a type of widely-expressed protein receptor on cell membranes, which can sense various signal molecules (such as hormones, neurotransmitters) from the extracellular environment and transduce these signals into intracellular space, thereby triggering a series of biological responses. They participate in a wide variety of physiological processes, including immune response, cell proliferation, and metabolic regulation. Recently GPCRs have become a hot research area for drug discovery and development[39].We identified the clusters inhibiting KF and KK formation based on the PPI network formed by the cluster-specific secretory proteins, GPCRs, and key hub genes that drive the formation of keloids. After that, we found 3 important clusters cluster0, cluster4 and cluster9 which can inhibit keloid formation by secreting proteins that can regulate hub genes (Fig. 5c-g). In cluster0 PPI network (Fig. 5d, e), these highly expressed secretory proteins are cluster0 markers can regulate the expression of the up hub gene IL1A and down hub gene JUN, FOS and PTGS2 in KK formation. The secretory proteins including COL1A1, FGF2, INHBA, IL6, VEGFA, IL1B, THBS1, TUFT1 and COL1A1 encodes the pro-alpha1 chains of type I collagen. Type I is a fibril-forming collagen found in most connective tissues and is abundant in bone, cornea, dermis and tendon. The FGF2 is a member of the fibroblast growth factor (FGF) family. FGF family members bind heparin and possess broad mitogenic and angiogenic activities. This protein has been implicated in diverse biological processes, such as limb and nervous system development, wound healing, and tumor growth. The INHBA encodes a member of the TGF-beta (transforming growth factor-beta) superfamily of proteins. The encoded preproprotein is proteolytically processed to generate a subunit of the dimeric activin and inhibin protein complexes. IL6, encodes a cytokine that functions in inflammation and the maturation of B cells. The protein is primarily produced at sites of acute and chronic inflammation, where it is secreted into the serum and induces a transcriptional inflammatory response through interleukin 6 receptor alpha. The functioning of this gene is implicated in a wide variety of inflammation-associated disease states. The VEGFA is a member of the PDGF/VEGF growth factor family; this growth factor induces proliferation and migration of vascular endothelial cells, and is essential for both physiological and pathological angiogenesis. Disruption of this gene in mice resulted in abnormal embryonic blood vessel formation. IL1B, The protein encoded by this gene is a member of the interleukin 1 cytokine family. This cytokine is produced by activated macrophages as a proprotein, this cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. THBS1, This protein is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. This protein has been shown to play roles in platelet aggregation, angiogenesis, and tumorigenesis. The TUFT1 has a function to be involved with adaptation to hypoxia, mesenchymal stem cell function, and neurotrophin nerve growth factor mediated neuronal differentiation. In conclusion, cluster0 express many secretory proteins to form a subtle chemotactic network in vivo for guiding circulating cells to the injury sites, play an important role in inflammation, angiogenesis and wound healing to inhibit the formation of KK. In cluster9 PPI network (Fig. 5c), these highly expressed secretory proteins are cluster9 markers can regulate the expression of the up hub gene IL6 in KF formation. The secretory proteins including GRN, LEPR, TRH, FGF9, FGF7 WNT2 GRN and Granulins are a family of secreted, glycosylated peptides, both the peptides and intact granulin protein regulate cell growth. Granulin family members are important in normal development, wound healing, and tumorigenesis. LEPR, This protein is a receptor for leptin, involved in the regulation of fat metabolism, as well as in a novel hematopoietic pathway that is required for normal lymphopoiesis. TRH, This gene encodes a member of the thyrotropin-releasing hormone family. Thyrotropin-releasing hormone is involved in the regulation and release of thyroid-stimulating hormone. FGF9, The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. This protein was isolated as a secreted factor that exhibits a growth-stimulating effect on cultured glial cells. FGF7, encoded protein is also a member of the fibroblast growth factor family. This protein is a potent epithelial cell-specific growth factor. Studies of mouse and rat homologs of this gene implicated roles in morphogenesis of epithelium, reepithelialisation of wounds, hair development and early lung organogenesis. WNT2, This gene is a member of the WNT gene family. The WNT gene family consists of structurally related genes which encode secreted signalling proteins. In conclusion, these secretory proteins regulate the up hub gene in KF formation by regulating the expression of GPCRs; these genes form a subtle chemotactic network in vivo play an important role in inflammation, cell growth, morphogenesis, tissue repair and wound healing to inhibit the formation of KF. Interestingly, In cluster4 PPI network(Fig. 5f-g), these highly expressed secretory proteins are cluster4 markers which can regulate the expression of the up hub gene IL6 in KF formation and IL1A in KK formation, secretory proteins including EGFR, HGF, IGFBP4, IGFBP5, IL1R1, IL6ST, NRP1, SFRP1, TGFBR3 and TIMP3. EGFR, The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor, thus inducing receptor dimerization and tyrosine autophosphorylation leading to cell proliferation. HGF, This gene encodes a protein that binds to the hepatocyte growth factor receptor to regulate cell growth, cell motility and morphogenesis in numerous cell and tissue types. This protein is secreted by mesenchymal cells and acts as a multi-functional cytokine on cells of mainly epithelial origin. This protein also plays a role in angiogenesis, tumorogenesis and tissue regeneration. IGFBP4, This gene is a member of the insulin-like growth factor binding protein (IGFBP) family, encodes a protein binds both insulin-like growth factors (IGFs) I and II and circulates in the plasma in both glycosylated and non-glycosylated forms. Binding of this protein prolongs the half-life of the IGFs and alters their interaction with cell surface receptors. IGFBP5, Enables insulin-like growth factor I binding activity involved in several processes, including cellular response to cAMP, regulation of smooth muscle cell migration and regulation of smooth muscle cell proliferation. The IL1R1 encodes a cytokine receptor that belongs to the interleukin-1 receptor family. The encoded protein is a receptor for interleukin-1 alpha, interleukin-1 beta, and interleukin-1 receptor antagonist. It is an important mediator involved in many cytokine-induced immune and inflammatory responses. IL6ST, The protein encoded by this gene is a signal transducer shared by many cytokines, including interleukin 6 (IL6), ciliary neurotrophic factor (CNTF). This protein functions as a part of the cytokine receptor complex. The activation of this protein is dependent upon the binding of cytokines to their receptors. Knockout studies in mice suggest that this gene plays a critical role in regulating myocyte apoptosis. NRP1, This gene encodes one of two neuropilins, which contain specific protein domains which allow them to participate in several different types of signaling pathways that control cell migration. Neuropilins bind many ligands and various types of co-receptors; they affect cell survival, migration, and attraction. Some of the ligands and co-receptors bound by neuropilins are vascular endothelial growth factor (VEGF) and semaphorin family members. TGFBR3, This locus encodes the transforming growth factor (TGF)-beta type III receptor. The encoded receptor is a membrane proteoglycan that often functions as a co-receptor with other TGF-beta receptor superfamily members. TIMP3, This gene belongs to the TIMP gene family. The proteins encoded by this gene family are inhibitors of the matrix metalloproteinases, a group of peptidases involved in degradation of the extracellular matrix (ECM). Expression of this gene is induced in response to mitogenic stimulation and this netrin domain-containing protein is localized to the ECM. These genes play important roles in immune regulation, organ development and control of cell growth. In conclusion, cluster4 express many secretory proteins. They can interact with GPCRs and hub genes to form a subtle chemotactic network in vivo for guiding circulating cells to the injury sites, and play an important role in inflammation, angiogenesis, cell migration and cell proliferation to inhibit the formation of KF and KK.
Validation of efficacy of identified therapeutic ADSC clusters in vivo
We first analyzed therapeutic clusters of the Minipigs ADSCs. The pADSCs-1 and pADSCs-2 samples donated by two miniature pigs were conducted the scRNA-seq experiment. In total, 11790 and 12539 cells were obtained respectively after the removal of low-quality cells by filtration. Data processing and clustering analysis were executed using the Seurat package (See method). The results showed that the pADSCs could be divided into 13 clusters and therapeutic clusters were identified in pADSC samples (Fig. 6a). The clustering analysis also showed significant consistency among the two replicates, demonstrating the reliability of the clustering method (Additional file 1: Fig. S6). The proportion of each pADSC cluster in total cells of two samples was calculated and compared (Fig. 6c).
We performed marker gene identification for 13 clusters, with the screening criteria for marker genes in clusters being adjusted P < .05 and |log2fc| >0.5 and we obtained top 300 marker genes in each cluster. We identified the therapeutic pADSC clusters according to the method for identifying hADSCs with therapeutic potential as described earlier. Finally, we found that cluster0, cluster1 and cluster9 probably can inhibit the keloid formation in Minipigs. In Cluster0, contains four different secretory proteins, GRN, TGFBR3, IGFBP5 and TIMP3, which can regulate the expression of KF hub genes by interacting with GPCRs, Involved in several processes, playing an important role in immune regulation, wound healing, organ development and control of cell growth, We infer that cluster0 is a key cluster for inhibiting KF. In Cluster1, contains four different secretory proteins, COL1A1, IL6ST, NRP1 and THBS1, which can regulate the expression of KK hub genes by interacting with GPCRs, Involved in several processes, playing an important role in cell migration, angiogenesis and control of cell growth, We infer that cluster1 is a key cluster for inhibiting KK. In Cluster9, contains four different secretory proteins, GRN, SFRP1, TIMP3 and THBS1, which can regulate the expression of both KF and KK hub genes by interacting with GPCR, Involved in several processes, playing an important role in normal development, wound healing, immune regulation, angiogenesis and control of cell growth, We infer that cluster9 is a key cluster for inhibiting KF and KK.
pADSCs with more therapeutic cluster cells have a better therapeutic effect on keloids
A total of 3 Bama minipigs were used in the study of keloid of full-thickness skin lesions. Each pig was surgically incised to create three 3 * 3 cm full-thickness defects on its back. One lesion was treated with sterile gauze as control (untreated), the other was treated with pADSCs-1 suspension (pADSCs-1), and the third was treated with pADSCs-2 suspension (pADSCs-2), all lesions covered with sterile gauze. The healing process of the wounds on the back of all pigs was consistent during the observation period. Scabs formation starting on day 4 and keloid on day 42 were observed on the untreated lesion. The lesion treated with pADSCs-2 displayed a much-accelerated healing process, although a complete keloid was achieved at the same time point (Fig. 7a, b).
Further analyses of the keloid by Masson stain and Sirius Red stain revealed that there was massive fibrous deposition across the healed region in the untreated group (Fig. 7a, c). In the pADSCs-1 treatment group, scar repair was still the main repair of full-layer skin defect, and only a small amount of normal skin tissue was observed near the boundary of uninjured tissue. However, in the pADSCs-2 suspension treated lesions, dispersed net-like deposition of fibrous that are similar to normal skin were more easily found in the regenerated area. Moreover, the fibrotic area of untreated control group was more than 30 mm2. Compared with the untreated control group, the fibrotic area in the pADSCs-1 suspension treatment group was slightly reduced, while the fibrotic area in the pADSCs-2 suspension treatment group was reduced by more than 30%, with an area less than 20 mm2 (Fig. 7c). The epithelization rate was also much more predominant in the lesions treated with pADSCs-2 suspension, which was more than 60% of the healed lesions covered by epithelium (Fig. 7d).