Method for promoting human pig cell xenochimerism and application thereof

Abstract

The application discloses a method for promoting human-pig cell xenogeneic chimerism and application thereof. The method comprises changing the expression amount of an HSPA5 gene in cells, wherein the expression amount of the HSPA5 gene in the cells is changed by overexpressing the expression amount of the HSPA5 gene in the cells. The application research finds that after hMSCs are injected into a pig embryo, HSPA5 overexpression can effectively improve the early survival efficiency of human cells in a xenogeneic embryo microenvironment, and it is proved that HSPA5 overexpression can effectively improve the survival ability of hMSCs in a xenogeneic embryo environment in vitro, and significantly improve the xenotransplantation efficiency of human cells, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for promoting human-pig cell chimerism and its application. Background Technology

[0002] Organ transplantation worldwide faces a severe shortage of donor organs. Current main solutions include xenotransplantation, interspecies chimaeras, and organoids. Interspecies chimaeras involves injecting pluripotent stem cells (PSCs, such as human stem cells) from one animal into the embryo of another, allowing the cells from one animal (e.g., human) to develop within the other animal and generate human organs, thus addressing the donor shortage.

[0003] Xenochimerism can facilitate the generation of human organs and tissues, the design of new drug screening methods, and the development of new human disease models. While interspecies blastocyst complementarity has been achieved between closely related species, such as mice and rats, this becomes more challenging for species that are far apart on the evolutionary tree. Due to the high degree of similarity between pigs, especially miniature pigs, and humans in many physiological, biochemical, and structural aspects, xenochimerism using pigs has become a research hotspot. In 2017, some studies successfully obtained human-pig chimeric embryos via microinjection, but the level and efficiency of chimerism were very low (Chinese patent application 201710601104.1). Figure 5 (Results). In light of this, researchers attempted to improve the survival rate of human cells in pigs through gene editing, including overexpression of the anti-apoptotic factor BCL2, mutation of the TP53 gene, and combined overexpression of the MYCN and BCL2 genes. This indicates that regulating gene expression can improve the survival rate of human cells in pig embryos, making it possible to generate humanized primitive organs in pigs with organogenesis disorders.

[0004] Humanized pig organs refer to pig organs that, through various technological means, contain human cells or possess certain characteristics of human organs, thus holding the promise of being used for human organ transplantation to address the organ shortage problem. It is worth noting that the normal internal temperature of the human body fluctuates between 36°C and 37°C, while the normal physiological temperature of pigs is 38°C to 39.5°C. Cell chimerism technology requires the injection of human stem cells into pig embryos through micromanipulation. Because increased environmental temperature causes human cells to be in a stress state, the chimerism efficiency is low. Therefore, methods to improve the chimerism efficiency of human-pig cells and promote the survival of humanized organs in pigs require further research.

[0005] Heat stress refers to the temperature stress experienced by cells or organisms when exposed to temperatures higher than their optimal growth temperature. It can trigger organelle localization disorders and interfere with the normal processes of intracellular synthesis and transport. Typical characteristics include a series of structural and functional damages to cells, such as endoplasmic reticulum and Golgi apparatus disruption, reduced numbers of mitochondria and lysosomes, and abnormal nucleolus and ribosome function. The endoplasmic reticulum, as a core organelle of eukaryotic cells, not only participates in lipid synthesis but also plays a crucial regulatory role in protein synthesis, modification, folding, and maturation, serving as an important structural basis for maintaining overall cellular homeostasis. Under heat stress, the normal folding process of intracellular proteins is hindered, leading to a large accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen, thereby activating the unfolded protein response (UPR).

[0006] UPR has a typical bidirectional effect on the regulation of cellular physiological homeostasis. Moderate endoplasmic reticulum stress can maintain normal cellular physiological function by clearing misfolded proteins and restoring endoplasmic reticulum homeostasis. If the stress is prolonged and exceeds the cell's compensatory capacity, UPR will change from a protective signal to an apoptotic signal, exerting a negative regulatory effect, and eventually leading to cell dysfunction or even death. Its main regulatory pathways are as follows: (1) Protein kinase R-like endoplasmic reticulum kinase (PERK) inhibits overall protein translation by phosphorylating eukaryotic initiation factor 2α (eIF2α) and reduces the accumulation of newly formed unfolded proteins. Meanwhile, p-eIF2α can specifically promote the expression of transcription factor ATF4, and further activate CHOP to initiate the apoptosis program under continuous heat stress; (2) the inositol-requiring enzyme 1α (IRE1α) pathway, after being activated by autophosphorylation, performs unconventional splicing of the mRNA of downstream transcription factor XBP1 to generate the activated form sXBP1. After entering the nucleus, sXBP1 can upregulate molecular chaperones, protein folding enzymes and endoplasmic reticulum degradation-related genes, and enhance the efficiency of protein folding and misfolded protein clearance; (3) activate the transcription factor 6 (ATF6) pathway, activate the endoplasmic reticulum-related degradation pathway, and maintain the structural and functional stability of the endoplasmic reticulum by specifically degrading misfolded proteins through the proteasome.

[0007] Glucose-regulated protein 78 (GRP78, also known as Bip or HSPA5) is a member of the heat shock protein 70 family and a key regulator of endoplasmic reticulum stress response. This protein contains an N-terminal ATP-binding domain and a C-terminal substrate-binding domain, allowing it to bind to the hydrophobic regions of unfolded or misfolded proteins, inhibiting protein aggregation and promoting correct folding to maintain endoplasmic reticulum homeostasis. Currently, there are no reports of promoting heterochimerism through HSPA5 regulation.

[0008] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for promoting human-pig cell xenogeneic chimerism and its application. In this invention, "promoting human-pig cell xenogeneic chimerism" refers to HSPA5 enhancing the survival ability of human mesenchymal stem cells (hMSCs) at pig physiological temperatures, specifically referring to the effective improvement of short-term survival of hMSCs in pig embryos by HSPA5 overexpression.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a method for promoting human-pig cell chimerism, the method comprising altering the expression level of the HSPA5 gene in cells, wherein altering the expression level of the HSPA5 gene in cells is achieved by overexpressing the expression level of the HSPA5 gene in the cells. Preferably, the method is also a method for improving the heat stress resistance of human mesenchymal stem cells in xenogeneic embryonic in vitro culture.

[0011] A second aspect of the invention provides another method for promoting human-porcine cell xenogeneic chimerism, the method comprising altering the expression level of the HSPA5 protein in cells, wherein altering the expression level of the HSPA5 protein in cells is achieved by overexpressing the expression level of the HSPA5 protein in the cells. Preferably, this method is also a method for improving the heat stress resistance of human mesenchymal stem cells in xenogeneic embryonic in vitro culture.

[0012] Preferably, in any of the above methods, the method includes altering the expression level of the HSPA5 gene or protein in human stem cells, wherein the human stem cells are selected from human embryonic stem cells, human pluripotent stem cells, or human mesenchymal stem cells.

[0013] Preferably, any of the above methods includes the following step: chimerizing human stem cells overexpressing HSPA5 with a porcine embryo.

[0014] Preferably, any of the above methods specifically includes the following steps: constructing an HSPA5 overexpression vector, transfecting human stem cells to obtain an HSPA5 overexpression cell line, injecting the HSPA5 overexpression cell line into cleavage-stage pig embryos via microinjection, and then culturing them in vitro.

[0015] More preferably, the cleavage-stage pig embryo includes a 2-cell stage pig embryo, a 4-cell stage pig embryo, an 8-cell stage pig embryo, or a morula, and the in vitro culture conditions are cultured at 38.5~39℃ until the pig embryo develops to the blastocyst stage.

[0016] A third aspect of the present invention provides a recombinant cell obtained by overexpressing the expression level of the HSPA5 gene in human stem cells or the expression level of the HSPA5 protein in cells.

[0017] In some embodiments, the method for obtaining recombinant cells is as follows: constructing an HSPA5 overexpression vector, transfecting human stem cells to obtain an HSPA5 overexpression cell line, and so on; the human stem cells are selected from human embryonic stem cells, human pluripotent stem cells or human mesenchymal stem cells.

[0018] The fourth aspect of the present invention provides the application of the above-described method or recombinant cells in any of the following: (1) Promotes human-pig cell chimerism and improves chimerism efficiency; (2) Preparation of xenogeneic chimeras of human stem cells and pig embryos.

[0019] The reagent is used in the preparation of a kit for promoting human-pig cell chimerism, and the reagent is used to overexpress HSPA5 in human stem cells. Preferably, the reagent is selected from plasmids, bacteriophages, artificial chromosomes, kinases, or viruses.

[0020] The beneficial effects of this invention are as follows: This invention has found that after injecting hMSCs into pig embryos, HSPA5 overexpression can effectively improve the early survival efficiency of human cells in the xenogeneic embryonic microenvironment, confirming that HSPA5 overexpression can effectively enhance the survival ability of hMSCs in the in vitro xenogeneic embryonic environment and significantly improve the xenograft efficiency of human cells, which has important application value. Attached Figure Description

[0021] Figure 1 These are the results of GO enrichment analysis based on RNA-seq.

[0022] Figure 2 This is a network diagram of differentiation-related gene enrichment.

[0023] Figure 3 The results of RNA-seq analysis of hMSCs cultured at different temperatures are as follows: (A) KEGG pathway enrichment analysis by RNA-seq; (B) MA diagram of differentially expressed genes in hMSCs; (C) enrichment network diagram of HSPA5-related biological processes; (D) qPCR detection of the effect of 39℃ heat stress on HSPA5 expression level in hMSCs. P < 0.01.

[0024] Figure 4 This is the result of the expression of HSPA5 and apoptosis-related proteins during heat stress.

[0025] Figure 5 The effects of HSPA5 knockdown on the biological function of hMSCs were shown: (A) qPCR verification of the knockdown efficiency of HSPA5 in hMSCs; (B) Western blot analysis of apoptosis-related proteins after HSPA5 knockdown; (C) Effect of HSPA5 knockdown on ROS levels in hMSCs; (D) CCK-8 assay to detect the effect of HSPA5 knockdown on hMSC proliferation. P < 0.05; P < 0.01; , P<0.0001.

[0026] Figure 6 This study demonstrates how HSPA5 overexpression protects hMSCs from heat stress damage: (A) qPCR detection of HSPA5 mRNA expression level; (B) immunofluorescence staining detection of intracellular localization of HSPA5 protein; (C) Western blot detection of apoptosis-related protein expression in HSPA5-overexpressing hMSCs; (D) CCK-8 assay detection of proliferation capacity of HSPA5-overexpressing hMSCs; (E) Immunofluorescence staining with the proliferation marker Ki67; (F) Quantitative statistical analysis of the mean fluorescence intensity of Ki67 protein; (G) Relative mean fluorescence intensity of ROS in HSPA5-overexpressing hMSCs; (H) Relative MDA content in HSPA5-overexpressing hMSCs; (I) Schematic diagram of the mechanism by which HSPA5 regulates cell survival under heat stress. P < 0.05; P < 0.01.

[0027] Figure 7 The following diagram illustrates the in vitro maturation and microinjection of porcine oocytes: (A) In vitro maturation of porcine oocytes; (B) Porcine embryos that have developed to the 2-cell stage after parthenogenetic activation; (C) Microinjection of hMSCs into 2-cell stage porcine embryos; (D) Schematic diagram of embryo micromanipulation process.

[0028] Figure 8The study showed that overexpression of HSPA5 improved the survival rate of hMSCs in porcine embryos: (A) representative fluorescence microscopy images of hMSCs in porcine embryos; (B) immunofluorescence staining images of hMSCs in porcine embryos; (C) quantitative statistical analysis of the number of hMSCs in porcine embryos (blastocysts); (D) distribution of hMSCs in porcine embryos in proliferation, quiescence and apoptosis states. P < 0.05; P < 0.01. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] Unless otherwise specified, the methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials used are conventional reagents and materials in the art and can be obtained commercially.

[0031] Example 1 1 Experimental Methods 1.1 RNA sequencing Human mesenchymal stem cells (hMSCs) were resuspended using TRIzol reagent, and total RNA was extracted according to the manufacturer's instructions. RNA quality was assessed using an Agilent 5300 bioanalyzer, and RNA concentration and purity were measured using a NanoDrop ND-2000 spectrophotometer. RNA purification, cDNA synthesis, and sequencing library construction were all performed according to Illumina standard procedures, and 2×150 bp paired-end sequencing was performed on an Illumina Novaseq 6000 platform.

[0032] Raw reads were quality controlled and filtered using FASTP to remove adapter sequences, cleave terminal N bases, and discard low-quality reads (Qphred ≤ 20 bases, > 50%). The filtered reads were then aligned to the human reference genome (Homo sapiens GRCh38 release-109) using HISAT2. Differentially expressed genes were screened using the DESeq2 package in R software, with a screening threshold of P-value ≤ 0.05 and absolute fold change ≥ 2. GO and KEGG enrichment analyses were performed on the differentially expressed genes using the clusterProfiler package, with a corrected P-value ≤ 0.05 used as the significance threshold, and the enrichment results were visualized.

[0033] 1.2 Quantitative Real-Time PCR Table 1 qPCR primer sequences

[0034] 1.3 Western blot for protein immunoblotting Conventional method.

[0035] 1.4 Construction of shRNA knockdown vector Using pLKO.1-Puro (Addgene 10878) as the backbone, a pLKO.1-EGFP-Puro vector carrying an EGFP tag was constructed. The digested vector was ligated with an annealed shRNA fragment using T4 DNA ligase to prepare an HSPA5-targeting shRNA interference vector. The shRNA sequence is shown in Table 2.

[0036] Table 2 shRNA sequences

[0037] Transformation and identification of positive clones: Competent cells were removed from the -80 ℃ freezer and placed on ice. 5 μL of ligation product was added to 50 μL of thawed Trans1-T1 (CD501, TransGen) competent cells and gently mixed. The mixture was incubated on ice for 30 min, then heat-shocked at 42 ℃ for 1 min, followed by an ice incubation for 2 min. 1 mL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37 ℃ with a shaker at 200 rpm for 60 min. The mixture was centrifuged at 6000 rpm for 1 min, the supernatant was discarded, and the cells were resuspended in 100 μL of LB liquid medium. The cells were evenly spread on LB agar plates containing ampicillin (100 μg / mL) and incubated overnight at 37 ℃. Single clones were picked, and positive clones were identified by Sanger sequencing. After amplification of the positive bacterial culture, plasmids were extracted using an endotoxin-free plasmid extraction kit (DP118, TianGen). The concentration and purity were measured using a micro-spectrophotometer, and the plasmids were stored at -20 ℃.

[0038] 1.5 Construction of HSPA5 overexpression vector 1.5.1 HSPA5 CDS Region Amplification The human HSPA5 reference sequence (NM_005347) was obtained from the NCBI database. Its full-length CDS is 1965 bp (SEQ ID NO.2), encoding 654 amino acids (SEQ ID NO.1). Using pLenti-CMV-GFP (Addgene 17448) as the backbone, a recombinant plasmid pLenti-CMV-HSPA5-GFP stably overexpressing HSPA5 was constructed. Specific amplification primers containing restriction enzyme sites and kozak sequences were designed; sequence information is shown in Table 3.

[0039] Table 3 HSPA5 CDS cloning primers

[0040] In Table 3, lowercase letters represent homologous regions of the cloning vector, underlined lines represent restriction enzyme sites, bold text represents the kozak sequence, and italics represent primers for the HSPA5 CDS region.

[0041] Total RNA was extracted from human bone marrow mesenchymal stem cells, and cDNA was obtained by reverse transcription. This cDNA was then used as a template for PCR amplification to obtain the PCR product and the target fragment of HSPA5.

[0042] 1.5.2 Double digestion of the overexpression cloning vector The pLenti-CMV-GFP vector was double-digested using BamHI (FD0054, Thermo) and SalI (FD0644, Thermo).

[0043] 1.5.3 Homologous recombination linkage Seamless cloning and ligation were performed using the pEASY®-Basic Seamless Cloning and Assembly Kit (CU201, TransGen) to ligate the target fragment of HSPA5 to the enzyme-digested overexpression vector. The reaction premix was placed in a PCR instrument and incubated at 50 °C for 15 min. After the reaction, the mixture was immediately transferred to ice for cooling.

[0044] 1.5.4 Transformation and PCR Identification of Bacterial Cultures Transformed into competent E. coli cells, single clones were picked, cultured, and identified. Positive clones were further verified for sequence correctness using Sanger sequencing. Positive plasmids with confirmed sequencing results were amplified and cultured, and recombinant plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0045] 1.6 Plasmid Extraction Conventional method.

[0046] 1.7 Lentiviral Packaging Conventional method.

[0047] 1.8 Establishment of overexpression and knockdown cell lines Human bone marrow mesenchymal stem cells (source: Cyagen Biosciences, catalog number HUXMF-01001) to be transfected were seeded in 12-well plates. When the cell confluence reached 30-40%, the culture medium was discarded, and concentrated lentivirus and 5 μg / mL Polybrene staining reagent were added. After 12 h of infection, the viral solution was discarded, and the cells were replaced with complete culture medium for further culture to obtain HSPA5 overexpressing cell lines for subsequent microinjection into 2-cell stage porcine embryos.

[0048] 1.9 Cell sorting (1) Digest cells with 0.25% trypsin, centrifuge at 250 g for 5 min, discard the supernatant and resuspend the cells with FASC.

[0049] (2) After the cell suspension was filtered through a 70 μm sterile cell sieve, GFP-positive cells were sorted using a BD flow cytometer. The sorted positive cells were identified by both qPCR and Western blotting.

[0050] 1.10 Cell viability assay Conventional method.

[0051] 1.11 Immunofluorescence staining Conventional method.

[0052] 1.12 ROS staining Conventional method.

[0053] 1.13 MDA Detection Conventional method.

[0054] 1.14 In vitro culture of porcine oocytes Pig ovaries were collected from local slaughterhouses and placed in physiological saline supplemented with penicillin-streptomycin antibiotics, then transported back to the laboratory within 2 hours. Using a 10 mL sterile syringe, the contents of follicles with a diameter of 3-5 mm were aspirated from the surface of the ovary, allowing them to settle naturally to collect cumulus-oocyte complexes (COCs). The collected COCs were washed twice with PBS containing 10% FBS, and COCs encapsulated by at least three intact layers of cumulus cells were selected under a stereomicroscope for subsequent in vitro maturation culture. 200 COCs were placed in each 1 mL of in vitro maturation medium and cultured at 39°C for 22-24 hours. Afterward, they were transferred to hormone-free maturation medium and cultured for another 20 hours. The expulsion of the first polar body was then used to determine mature oocytes.

[0055] 1.15 Parthenogenetic activation of porcine oocytes Mature porcine oocytes were gently pipetted in 0.1% hyaluronidase solution to remove cumulus granulosa cells. The oocytes were washed three times with the manipulation solution, followed by equilibration in fusion medium for 1-2 min. Using an ECM2001 electrofusion instrument, the oocytes were electroactivated with a DC pulse of 1.2 kV / cm for 30 μs. After electroactivation, the oocytes were transferred to PZM culture droplets and cultured in a constant temperature incubator to obtain 2-cell stage embryos.

[0056] 1.16 Microinjection Take 20-30 2-cell stage embryos and place them in a preheated 20 μL droplet of manifold solution, then seal with paraffin oil. Using a microinjection apparatus, inject 3-4 hMSCs into the periocular space of the 2-cell stage porcine embryos. After injection, transfer the embryos to PZM culture medium droplets for further culture at 39°C until they reach the blastocyst stage, which takes 6-7 days, depending on the embryo's development. Collect the blastocysts for analysis.

[0057] 1.17 Immunofluorescence detection of porcine embryos (1) Use an oral pipette to transfer the desired embryos into an acidic saturated solution to digest and remove the zona pellucida.

[0058] (2) Wash three times with PBS-PVP, and fix with 4% PFA at room temperature in the dark for 30 min.

[0059] (3) Wash the embryos three times with PBS-PVP, place them in a 4-well plate, add 0.2% Triton X-100 and permeate at room temperature for 20 min.

[0060] (4) Block non-specific antigen binding sites in 1% BSA blocking solution at 37 ℃ for 30 min.

[0061] (5) Wash three times with PBS-PVP and incubate overnight with primary antibody at 4 ℃.

[0062] (6) Wash three times with PBS-PVP, add fluorescent secondary antibody, and incubate at 37 °C in the dark for 1 h.

[0063] (7) After washing three times with PBS-PVP, the embryo was transferred to Hoechst droplets and stained at room temperature in the dark for 20 min, and then washed thoroughly with PBS-PVP.

[0064] (8) Apply a mounting stick to each of the four corners of the slide, drop an anti-quenching mounting medium into the center, and transfer the embryo into the mounting medium. Cover with a coverslip and mount the slide. Acquire images using a laser confocal microscope.

[0065] 1.18 Statistical Methods Data are expressed as mean ± standard error, and experiments were performed in at least three biological replicates. Statistical analysis was performed using GraphPadPrism 9 software. Data were analyzed using t-tests, and p < 0.05 was considered statistically significant.

[0066] 2 Experimental Results 2.1 RNA-seq analysis revealed that porcine physiological temperature inhibits the expression of hMSCs differentiation-related genes. To further investigate the transcriptional regulatory mechanism of porcine physiological temperature on hMSCs, this study collected MSC-H and MSC-L cells for RNA sequencing (RNA-seq) analysis. Paired-end sequencing was performed using the Illumina platform, and subsequent differential expression analysis identified 1200 differentially expressed genes, including 651 upregulated genes and 549 downregulated genes. GO enrichment analysis showed that porcine physiological temperature significantly affects the osteogenic and chondrogenic differentiation processes of hMSCs and participates in regulating cholesterol biosynthesis pathways related to adipogenic differentiation. Figure 1 ).

[0067] Network analysis of differentiation-related gene enrichment showed that the expression of bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein receptor type 1B (BMPR1B), core regulators of osteogenic differentiation, was significantly downregulated; the expression level of peroxisome proliferator-activated receptor gamma (PPARG), a key regulator of adipogenic differentiation, was significantly reduced; and the expression of growth differentiation factor 5 (GDF5) was also significantly downregulated. Figure 2 This suggests that porcine physiological temperature may alter the differentiation potential of hMSCs by regulating the expression levels of the aforementioned key genes.

[0068] 2.2 RNA-seq screening and verification that HSPA5 is a core gene regulating hMSCs function at physiological temperature in pigs To further clarify the molecular mechanism by which porcine physiological temperature regulates the biological function of hMSCs, this study performed KEGG pathway enrichment analysis on RNA-seq data. The results showed that the proteasome-mediated protein degradation pathway was significantly downregulated, suggesting that porcine physiological temperature can disrupt protein homeostasis within hMSCs. Figure 3A). Based on existing research, it is speculated that this homeostasis imbalance is closely related to heat stress-induced protein misfolding and accumulation of unfolded proteins. Under physiological conditions, cells can mitigate UPR-induced cell damage by slowing down protein synthesis and upregulating molecular chaperone expression. Screening based on differentially expressed gene MA maps revealed an upregulated expression level of HSPA5, a core regulator of UPR. Figure 3 B). Meanwhile, GO enrichment analysis showed that HSPA5 plays a crucial regulatory role in key biological processes such as maintaining organelle protein localization and mediating unfolded protein responses. Figure 3 C). To verify the reliability of the RNA-seq results, qPCR was used to detect the expression level of HSPA5 after heat stress treatment at 39 ℃. The results showed that the relative expression level of HSPA5 mRNA in the MSC-H group was upregulated by 4 times compared with that in the MSC-L group (C). Figure 3 D).

[0069] Short-term heat stress at physiological porcine temperatures specifically induces the expression of HSPA5 (GRP78) in hMSCs, thereby initiating endogenous cellular protective mechanisms to maintain basal cellular homeostasis. During this stress adaptation phase, the endoplasmic reticulum stress-related PERK-eIF2α-ATF4 pathway is not fully activated, and the expression of anti-apoptotic proteins Bcl-2 and pro-apoptotic proteins Bax and CHOP shows no significant changes, suggesting that the upregulation of HSPA5 under short-term heat stress can effectively resist stress damage and prevent cells from entering the apoptotic program. As the duration of heat stress increases, the expression level of HSPA5 in hMSCs gradually declines, and its protective regulatory effect weakens accordingly, thereby activating the PERK-eIF2α-ATF4 pathway, leading to a decrease in Bcl-2 expression and a significant increase in Bax and CHOP expression, resulting in cells entering an apoptotic state. Figure 4 ).

[0070] 2.3 Knockdown of HSPA5 promotes apoptosis of hMSCs and exacerbates cellular oxidative stress. To clarify the core regulatory role of HSPA5 in hMSCs responding to the 39 °C heat stress microenvironment, this study used pLKO.1-EGFP-Puro as the vector backbone and constructed a recombinant shRNA interference vector targeting the HSPA5 gene using molecular cloning technology. The successful construction of the vector was verified by Sanger sequencing. Lentiviral-mediated gene transfection was used to introduce the interference plasmid into hMSCs, and high-purity GFP-positive stable transfected cell lines were obtained by flow cytometry sorting. qPCR and Western blotting results showed that, compared with the control group, the mRNA transcription level and protein level of HSPA5 in the shHSPA5-1 and shHSPA5-2 groups were significantly downregulated, fully confirming the successful construction of a stably knocked-down hMSCs cell model, which can be used for subsequent functional experiments. Figure 5A).

[0071] Western blot analysis showed that knocking down HSPA5 activated the key endoplasmic reticulum stress pathway PERK-eIF2α-ATF4 in hMSCs, while downregulating the expression of the anti-apoptotic protein Bcl-2 and significantly upregulating the expression of the pro-apoptotic protein Bax, suggesting that decreased HSPA5 expression can enhance the cell apoptosis tendency. This phenotype is consistent with the apoptosis results of hMSCs induced by long-term heat stress. Figure 5 B). ROS fluorescence detection results showed that, compared with the control group, the HSPA5 knockdown group had increased intracellular ROS fluorescence intensity and aggravated intracellular oxidative stress. Figure 5 C). Furthermore, CCK-8 proliferation assay results showed that knocking down HSPA5 mildly inhibited the in vitro proliferation activity of hMSCs (C). Figure 5 D). The above results suggest that HSPA5 mainly participates in the response of hMSCs to 39 °C heat stress by regulating the PERK-eIF2α-ATF4 pathway, thereby affecting cell apoptosis, oxidative stress and proliferation.

[0072] 2.4 Overexpression of HSPA5 enhances the heat tolerance of hMSCs To further verify the core regulatory function of HSPA5 in the response of hMSCs to 39 °C heat stress, this study constructed an HSPA5 overexpression vector using pLenti-CMV-GFP as the backbone. Using hMSCs cDNA as a template, the complete coding region of HSPA5 was amplified by PCR. Agarose gel electrophoresis showed that the target fragment was approximately 2000 bp in size, consistent with expectations. The pLenti-CMV-GFP vector was linearized by double digestion with BamHI and SalI, and the linearized vector backbone fragment was recovered. The HSPA5 coding region was ligated to the linearized vector using homologous recombination technology. After transformation into competent cells, single colonies were selected and amplified. Preliminary identification by bacterial PCR and verification by Sanger sequencing confirmed the successful construction of the pLenti-CMV-HSPA5-GFP overexpression vector.

[0073] Using a lentivirus-mediated gene delivery system, the successfully constructed pLenti-CMV-HSPA5-GFP recombinant plasmid was transfected into hMSCs. GFP-positive cells were enriched by flow cytometry to obtain a cell line stably overexpressing HSPA5. The gene overexpression efficiency was validated using qPCR and Western blotting. The results showed that HSPA5 was significantly highly expressed at both the mRNA transcriptional and protein levels in the overexpression group, indicating that the overexpression cell model was successfully constructed. Figure 6 A, C). Further immunofluorescence staining combined with confocal microscopy revealed that the overexpressed HSPA5 protein (GRP78) was mainly enriched in the cytoplasm and cell membrane regions. Figure 6 B). Notably, HSPA5 overexpression significantly inhibited heat stress-induced abnormal activation of the PERK-eIF2α-ATF4 endoplasmic reticulum stress pathway, while simultaneously upregulating the expression level of the anti-apoptotic protein Bcl-2, thus enhancing the cell's anti-apoptotic ability. Figure 6 C). Importantly, CCK-8 cell proliferation assays and Ki67 immunofluorescence staining results showed that, compared to the blank control group, HSPA5 overexpression significantly promoted the proliferation of hMSCs. Figure 6 DF). Furthermore, HSPA5 overexpression can alleviate oxidative stress damage and maintain cellular redox homeostasis by reducing intracellular ROS and MDA levels. Figure 6 GH). The above results indicate that HSPA5 overexpression can effectively alleviate heat stress-induced cell damage by promoting hMSC proliferation, enhancing their antioxidant and anti-apoptotic capabilities, and thus improving the cell's heat tolerance. Figure 6 I).

[0074] 2.5 Overexpression of HSPA5 improves the survival efficiency of hMSCs in porcine embryos After xenotransplantation of human cells into pigs, immune rejection is highly likely to occur, making it difficult to accurately determine whether HSPA5 overexpression can effectively enhance the heat tolerance of human cells. Considering that pig embryos in an in vitro culture system can stably simulate the normal physiological temperature environment in pigs while effectively avoiding interference from immune rejection factors, this study selected in vitro cultured pig embryos as an experimental model to conduct subsequent related experiments. The contents of follicles with a diameter of 3-5 mm from the pig ovary were collected using a 10 mL syringe and cultured in an in vitro culture system for 42-44 h, successfully obtaining mature oocytes. Figure 7 A). Subsequently, granulosa cells surrounding the oocytes were removed by hyaluronidase digestion, and embryonic development was induced through parthenogenetic activation to obtain 2-cell stage porcine embryos. Figure 7 B). Gene-edited hMSCs were injected into 2-cell stage porcine embryos using microinjection to investigate the survival of HSPA5-overexpressing hMSCs in porcine embryos. Figure 7 CD).

[0075] After injection, the embryos developed to the blastocyst stage, and were collected for subsequent testing and analysis. Fluorescence microscopy observations showed that exogenous hMSCs successfully colonized the pig embryos. Figure 8 A). In this study, 3-4 hMSCs were injected into 2-cell stage porcine embryos, and the hMSCs in the blastocysts were quantitatively analyzed using immunofluorescence staining. The results showed that compared with the control group (injected with hMSCs that did not overexpress HSPA5), the number of hMSCs overexpressing HSPA5 in porcine embryos was significantly increased. Figure 8 B). Cell viability was classified based on cell count: fewer than 3 hMSCs were considered apoptotic, 3-4 were considered quiescent, and more than 4 were considered proliferative. Further analysis based on this criterion showed that in the control group, 62% of hMSCs were quiescent, 11% were apoptotic, and only 27% were proliferative; while in the experimental group, 74% of HSPA5-overexpressing hMSCs were proliferative. Figure 8 CD).

[0076] The above results indicate that HSPA5 overexpression can promote the survival of hMSCs in porcine embryos, thereby improving the efficiency of xenografting of human cells.

[0077] 3. Summary and Analysis The results of this invention show that: (1) By RNA-seq analysis, we screened and verified that HSPA5, a key regulator of unfolded protein response, was upregulated in hMSCs under heat stress, confirming that it is the core gene for maintaining hMSC homeostasis under short-term heat stress.

[0078] (2) HSPA5 knockdown can significantly activate the PERK-eIF2α-ATF4 signaling pathway in hMSCs, exacerbate apoptosis, increase intracellular oxidative stress, and mildly inhibit cell proliferation.

[0079] (3) HSPA5 overexpression can enhance the anti-apoptotic, anti-oxidative stress and proliferation capacity of hMSCs by inhibiting the PERK-eIF2α-ATF4 signaling pathway.

[0080] (4) HSPA5 overexpression can effectively improve the short-term survival of hMSCs in pig embryos.

[0081] The heat shock response is a highly conserved adaptive protective response that cells rapidly initiate under various stress stimuli such as heat stress, oxidative stress, and hypoxia. Its core characteristic is the significant induction of HSP expression, which repairs denatured proteins, maintains protein homeostasis and cellular structural stability through molecular chaperone functions, thereby enhancing cellular tolerance and survival. Based on molecular weight, HSPs can be classified into seven families: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, small heat shock proteins, and ubiquitin. Studies have confirmed that HSPA5, a member of the Hsp70 family, is a core regulatory molecule of the endoplasmic reticulum (UPR). When cells are at homeostasis, GRP78 binds in an inactive form to the transmembrane stress sensors ATF6, PERK, and IRE1 in the UPR pathway. When unfolded proteins accumulate in large quantities in the endoplasmic reticulum, GRP78 dissociates from these sensors and regulates cell proliferation, oxidative stress tolerance, and apoptosis through multiple signaling pathways.

[0082] Our results indicate that short-term heat stress can enable hMSCs to maintain intracellular homeostasis and exert a cytoprotective effect by upregulating HSPA5 expression. With prolonged heat stress, HSPA5 expression gradually declines, weakening its protective effect and subsequently activating the PERK-eIF2α-ATF4 signaling pathway, ultimately inducing hMSC apoptosis. HSPA5 gene knockdown experiments showed significant activation of the PERK-eIF2α-ATF4 pathway in hMSCs, with a markedly enhanced apoptotic tendency, consistent with the cell state after long-term heat stress treatment. Notably, HSPA5 overexpression significantly inhibited PERK-eIF2α-ATF4 pathway activation and upregulated the expression of the anti-apoptotic protein Bcl-2, effectively alleviating heat stress-induced damage to hMSCs. CCK-8 proliferation assays and Ki67 immunofluorescence staining results showed that HSPA5 overexpression enhanced the proliferation capacity of hMSCs. Consistent with the above results, previous studies have confirmed that HSPA5 plays a key role in maintaining cell survival and can promote the proliferation of various cell types, including synovial cells, osteosarcoma cells, and prostate cancer cells.

[0083] Xenotransplantation of human cells into pigs is a crucial technological pathway for constructing functional humanized organs, potentially alleviating the shortage of clinical organ donors. Wu et al. successfully constructed human-pig chimeric embryos by microinjecting human PSCs into pig embryos. However, compared to human-mouse cell chimerism, the current level and integration efficiency of human-pig cell chimerism remain generally low, becoming a key bottleneck restricting the development of this technology. Currently, researchers are using strategies such as gene editing and optimization of embryo culture systems to improve the survival and integration efficiency of human cells in pigs. Previous studies have successfully obtained humanized endothelial cells, skeletal muscle tissue, and mid-stage kidney structures in pig models, demonstrating the feasibility of cultivating functional humanized organs in pig models with organogenesis defects. Considering the developmental characteristics of pig embryos, this study selected 2-cell stage pig embryos as an experimental model to systematically evaluate the survival ability of hMSCs in the xenogeneic embryonic microenvironment. The results showed that HSPA5-overexpressing hMSCs were injected into 2-cell stage pig embryos and cultured in vitro to the blastocyst stage, with a significantly higher cell survival rate than the unmodified control group hMSCs, providing a new candidate scheme for optimizing the efficiency of human cell xenotransplantation using gene editing.

[0084] Previous research in this invention used hMSCs and porcine embryos as experimental models to explore the effects of interspecies physiological temperature differences on the biological functions of human cells. The results showed that under culture conditions simulating porcine physiological temperature, hMSCs exhibited significant cell damage phenotypes, specifically accelerated cell senescence, reduced proliferation capacity, increased oxidative stress levels and early apoptosis rates, and decreased trilineage differentiation potential. This invention demonstrates that HSPA5 overexpression can enhance the anti-apoptotic, anti-oxidative stress, and proliferative capacity of hMSCs by targeting and inhibiting the PERK-eIF2α-ATF4 signaling pathway. More importantly, HSPA5 overexpression effectively improved the early survival efficiency of human cells in the xenogeneic embryonic microenvironment after injection of hMSCs into 2-cell stage porcine embryos. In summary, this study not only reveals the impact of interspecies physiological temperature differences on the biological characteristics of human cells but also confirms that HSPA5 overexpression can effectively improve the survival capacity of hMSCs in an in vitro xenogeneic embryonic environment, demonstrating potential application value.

[0085] The protein encoded by the human HSPA5 gene (SEQ ID NO.1): MKLSLVAAMLLLLSAARAEEEDKKEDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGRTWNDPSVQQDIKFLPFKVVEKKTKPYIQVDIGGGQTKTFAPEEISAMVLTKMKETAEAYLGKKVTHAVVTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNGVFEVVATNGDTHLGGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAVQKLRREVEKAKRALSSQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLDVCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYGSAGPPPTGEEDTAEKDEL.

[0086] Human HSPA5 gene CDS sequence (SEQ ID NO. 2):

Claims

1. A method for promoting human-pig cell chimerism, characterized in that, The method includes altering the expression level of the HSPA5 gene in cells, wherein altering the expression level of the HSPA5 gene in cells is achieved by overexpressing the expression level of the HSPA5 gene in the cells.

2. A method for promoting human-pig cell chimerism, characterized in that, The method includes altering the expression level of HSPA5 protein in cells, wherein altering the expression level of HSPA5 protein in cells is achieved by overexpressing the expression level of HSPA5 protein in the cells.

3. The method according to claim 1 or 2, characterized in that, The method includes altering the expression level of the HSPA5 gene or protein in human stem cells, wherein the human stem cells are selected from human embryonic stem cells, human pluripotent stem cells, or human mesenchymal stem cells.

4. The method according to claim 3, characterized in that, The process includes the following steps: chimerizing human stem cells overexpressing HSPA5 with pig embryos.

5. The method according to claim 4, characterized in that, The process includes the following steps: constructing an HSPA5 overexpression vector, transfecting human stem cells to obtain an HSPA5 overexpression cell line, injecting the HSPA5 overexpression cell line into cleavage-stage pig embryos via microinjection, and then culturing them in vitro.

6. The method according to claim 5, characterized in that, The cleavage-stage pig embryos include 2-cell stage pig embryos, 4-cell stage pig embryos, 8-cell stage pig embryos, or morula embryos. The in vitro culture conditions are 38.5~39℃ until the pig embryos develop to the blastocyst stage.

7. A recombinant cell, characterized in that, The recombinant cells were obtained by overexpressing the HSPA5 gene or the HSPA5 protein in human stem cells.

8. The recombinant cell according to claim 7, characterized in that, The method for obtaining recombinant cells is as follows: construct an HSPA5 overexpression vector, transfect human stem cells to obtain an HSPA5 overexpression cell line, and thus obtain the recombinant cells; The human stem cells are selected from human embryonic stem cells, human pluripotent stem cells, or human mesenchymal stem cells.

9. The use of the method of claim 1 or 2 or the recombinant cells of claim 7 in any of the following: (1) Promotes human-pig cell chimerism and improves chimerism efficiency; (2) Preparation of xenogeneic chimeras of human stem cells and pig embryos.

10. Application of the reagent in the preparation of the kit, the kit being used to promote human-pig cell xenogeneic chimerism, the reagent being used to overexpress HSPA5 in human stem cells, preferably the reagent being selected from plasmids, bacteriophages, artificial chromosomes, kinases or viruses.

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