CN117126949B - Cell type and traceability method for identifying primary cells in amniotic fluid sample - Google Patents
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Abstract
The invention provides a method for identifying the cell type and tracing the primary cells in amniotic fluid samples, which is characterized by comprising the following steps: s1, capturing single cells in primary cells in a amniotic fluid sample, and sequencing to obtain data of the amniotic fluid sample; s2, fetal cell data are obtained; s3, grouping cells and carrying out cluster analysis; s4, carrying out high expression gene identity identification on the group without fetal cell distribution; s5, carrying out identification and annotation of organ sources and germ layers sources on amniotic fluid cells. The invention not only performs cell tracing aiming at amniotic fluid samples, but also explains the organ sources and germ layer sources of amniotic fluid cells, so that the classification and tracing of cells are more accurate.
Description
Technical Field
The invention relates to the field of medicine, in particular to a method for identifying cell types and tracing primary cells in amniotic fluid samples.
Background
Amniotic sac is a pair of tough but thin transparent membranes, the outer membrane is also called chorion, which surrounds the amniotic membrane, which is part of the placenta, and the inner membrane, i.e., the amniotic membrane, surrounds amniotic fluid and fetus. The initial amniotic fluid is isotonic, contains carbohydrates, proteins, phospholipids, lipids, urea, electrolytes, and the like, and changes its composition as urine discharged from the fetus increases. The amniotic fluid can keep consistent pressure and temperature, plays a role in buffering and protecting embryos, can allow the fetus to freely move in uterus, is beneficial to the musculoskeletal development and blood flow of the fetus, and in addition, the fetus can inhale the amniotic fluid to promote the normal growth and development of the lung, and the fetus can swallow the amniotic fluid in uterus to be beneficial to the development of the gastrointestinal tract. Amniotic fluid is 207+/-92 ml at 6 weeks of pregnancy, 258+/-97 ml at 18 weeks and 365+/-88 ml at 20 weeks, and the number of amniotic fluid cells is relatively heterogeneous, the number of cells in amniotic fluid in the middle of pregnancy varies between 10 and 1000 cells/μl, and most researches consider that amniotic fluid cells are composed of various fetal tissue shedding cells in contact with amniotic fluid, but the cell types of amniotic fluid cells and the number ratio of various cell types are still unknown.
At present ,"Single-Cell Transcriptomics of Cultured Amniotic Fluid Cells Reveals Complex Gene Expression Alterations in Human Fetuses With Trisomy 18", a cultured second-generation amniotic fluid cell sample is firstly collected, then single-cell transcriptome sequencing is carried out by using a 10xGenomics single-cell capturing and sequencing technology, after dimension-reduction clustering analysis is carried out on data by using t-SNE, the differential expression genes of cells in a specific group (Cluster) and all other groups (Cluster) of cells are firstly determined by using Wilcox in a Seurat package, and then the differential expression genes of cells in the specific group (Cluster) and all other groups (Cluster) of cells are determined by using a DESeq2 package. However, this method did not conduct studies on lineage and germ layer traceability, but the amniotic fluid cells were classified into 6 major classes according to functional analysis.
"scRNA-Seq of Cultured Human Amniotic Fluid from Fetuses with Spina Bifida Reveals the Origin and Heterogeneity of the Cellular Content" Firstly, amniotic fluid cells are subjected to in vitro culture and adherence for 5 days, namely P0 generation cells are collected, then single cell transcriptome sequencing is carried out by using a 10xGenomics single cell capturing and sequencing technology, data are subjected to dimension reduction clustering analysis by using t-SNE, the average expression difference between cells in a test group (Cluster) and other cells is at least 0.25 times (logarithmic scale), the genes with the P value of less than 0.05 after adjustment are identified as differential expression genes, and then enrichment analysis is carried out by using an R packet clusterProfiler, namely the differential expression genes of each group (Cluster) are selected and analyzed according to biological processes in Gene Ontology as categories. Finally, the study identified 4 tissue sources of amniotic fluid cells by high expression genes, and then identified different cell types of the same tissue sources by high expression genes, but no analysis of germ layer sources was performed in the method, and the sources of the cells were not known.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for identifying the cell type and tracing the primary cells in the amniotic fluid sample, and the method is used for identifying the cell identity of the primary cells without culturing the cells in the process of identifying the cell identity, so that the cell screening caused by the culturing process is avoided, the change of the cell type or the cell number in the culturing process is also avoided, and the analysis of the primary cells in the amniotic fluid sample is more accurate by classifying the amniotic fluid cells and identifying the cell identity through high-expression genes.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
a method for identifying the cell type and tracing of primary cells in amniotic fluid samples, comprising the steps of;
s1, capturing single cells in primary cells in a amniotic fluid sample, and sequencing to obtain data of the amniotic fluid sample;
s2, fetal cell data are obtained;
S3, grouping cells and carrying out cluster analysis;
s4, carrying out high expression gene identity identification on the group without fetal cell distribution;
S5, carrying out identification and annotation of organ sources and germ layers sources on amniotic fluid cells.
Further, the specific steps of capturing single cells and sequencing in the step S1 are as follows: single cell capturing is completed through a single cell capturing platform; the mRNA is then captured for reverse transcription and library-building sequencing.
Further, the specific steps of obtaining fetal cell data in S2 are: and collecting gene expression matrixes of single-cell transcriptome data of the fetus, and cell type and organ source information of each cell.
Further, the gestational age of the fetus is close to the gestational week of the amniotic fluid sample to be analyzed.
Further, the specific steps of S3 cell grouping are as follows:
1) Respectively constructing Seruat objects from fetal cell data and amniotic fluid sample data, wherein the sample data and the fetal cell data are mapped;
2) Separating cells into different groups (Cluster) by UMAP Cluster analysis;
3) The number of cells of different cell types of fetal data and amniotic fluid sample cells distributed in different groups (Cluster) was counted, and the most distributed fetal cell type in each group (Cluster) was identified as the cell type of that group (Cluster).
Further, the plurality of cell types obtained in the 3) can be identified, wherein the group with fetal cell distribution can be identified, and the group without fetal cell distribution can be identified by high expression genes.
Further, the specific steps of the identification of the S4 high expression gene are as follows:
1) FINDALLMARKER was used to find highly expressed genes in a Cluster (Cluster) without fetal cell distribution;
2) Screening high-expression genes of a group (Cluster) without fetal cell distribution, and setting filtering and screening conditions;
3) Cell identity was performed on highly expressed genes of a Cluster (Cluster) without fetal cell distribution using CELLMARKER database.
Further, the specific steps of annotating the amniotic fluid cells with organ sources and germ layer sources in S5 are as follows: based on organ origin, germ layer origin was determined.
The invention also provides the application of the method in identifying the cell type and tracing of primary cells in amniotic fluid samples, wherein the application is for non-diagnosis and treatment purposes.
The invention has the beneficial effects that: the method of the invention carries out cell identity identification aiming at primary cells in amniotic fluid samples, and the cells are not cultured, thereby avoiding 'cell screening' caused by the culture process and avoiding the change of cell types or numbers in the culture process. According to the invention, fetal cell data are referred to for tracing amniotic fluid cells in similar gestational weeks, so that not only is the amniotic fluid cells derived from a fetus demonstrated, but also the amniotic fluid cells are sample information in similar gestational weeks, and the tracing of the amniotic fluid cells is more reliable. According to the invention, cell tracing is performed on the amniotic fluid sample, the organ source and the germ layer source of the amniotic fluid cells are described, and cell identity identification is performed on specific cell types through high expression genes, so that the classification and tracing of the cells are more accurate.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a schematic representation of the expression of fetal cells and amniotic fluid cells in different populations (Cluster);
FIG. 2 is a schematic representation of cell types of each Cluster;
FIG. 3 is a schematic diagram of amniotic fluid cell types and cell numbers.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1:
This example is a single cell in 8 mid-gestation amniotic fluid samples. The aim was to determine the identity of these single cells and thus explore the organ and germ layer origin of amniotic fluid cells, and a total of 30063 single cells were obtained from 8 amniotic fluid samples of this batch. The method for identifying the cell type and tracing the primary cells in the amniotic fluid sample comprises the following steps:
s1, single cell capture sequencing
The single cell capturing is completed by carrying out the process through a BD Rhapsody platform, firstly preparing the cells into a specific single cell suspension, and randomly distributing the single cells into more than 200,000 micropores according to a limiting dilution method. Magnetic beads were then added to capture mRNA and reverse transcribed, and pool sequencing was performed.
S2, selecting cell information of reference data
Single cell transcriptome data of mid-gestation (10-18 weeks) fetuses were selected and stored in the loom format. Firstly, collecting a gene expression matrix of cells and cell type and organ source information of each cell by using loomR bags, and taking 1:100 as a fixed interval, namely taking data information of 1 cell every 100 cells, taking data information of 40621 cells in total, and carrying out subsequent integrated analysis to obtain fetal cell data.
S3, cell grouping
The data of fetal cell data and 8 amniotic fluid samples are respectively constructed to Seruat objects, wherein the 8 sample data are combined and then mapped with the fetal cell data, the cells are divided into 22 groups (clusters) through UMAP Cluster analysis (resolution=0.4), then the different cell types of the fetal data and the cell numbers of the amniotic fluid samples distributed in the different groups (clusters) are counted, the fetal cell type most distributed in each group (Cluster) is identified as the cell type of the group (Cluster), and 18 cell types are identified in total, as shown in fig. 1.
Obtaining inhibitory neurons of the brain from group 0 (Cluster 0), the brain from group 1 (Cluster 1) and group 5 (Cluster 5), the brain from excitatory neurons, the cerebellum from group 3 (Cluster 3), the cerebellum from group 11 (Cluster 11), the cerebellum from group 2 (Cluster 2) and the adrenal cortical cells from group 4 (Cluster 4), the liver from group 6 (Cluster 6), the liver from group 9 (Cluster 9) and group 16 (Cluster 16), the pancreas from group 7 (Cluster 7), the lung from group 8 (Cluster 8), the lung from group 10 (Cluster 10), the different organ-derived vascular endothelial cells from group 13 (Cluster 11), the different organ-derived vascular endothelial cells from group 14 (Cluster 14), the different organ-derived stromal cells from group 15 (Cluster) and the liver from group 21, the liver from group 21 (Cluster) and the main brain from group 21, the liver from group 21 and the liver from group 21. While group 12 (Cluster 12) and group 18 (Cluster 18) still require specific high-expression genes for identification.
S4, carrying out identity authentication on the high-expression gene
The high expression genes in various groups (Cluster) are found by FINDALLMARKER, then the found high expression genes in all groups (Cluster) are screened, the top50 is extracted, the filtering screening condition (avg_ logFC > =2, pct.1> =0.4 and pct.2> =0.4) is set, the high expression genes in group 18 (Cluster 18) are CSF3R, CXR2 and SELL, and then cell identity identification is carried out on the high expression genes in group 18 (Cluster 18) by using CELLMARKER database (http:// 117.50.127.228/CELLMARKER/index.html), wherein CSF3R and CXR2 are high expressed in neutrophils, SELL is high expressed in neutrophils and T cells, and group 18 (Cluster 18) is neutrophils. Since there is no high expression gene of Cluster12 (Cluster 12) after filtering and screening, the high expression gene of top3 of Cluster12 (Cluster 12) found by FINDALLMARKER is identified. The high expression genes of the group 12 (Cluster 12) are CD74, C1QC and CTSB, wherein the CD74 and the CTSB are highly expressed in microglia of the brain, the C1QC is expressed in medullary cells of the brain, and the group 12 (Cluster 12) is consistent with the group 19 (Cluster 19), namely microglia.
In summary, each cell group was annotated according to fetal database and high-expression genes, and as shown in fig. 2, 17 cell types were identified in total.
S5, amniotic fluid cell identity identification and organ source and germ layer annotation
Of the 17 cell types identified, amniotic fluid cells were predominantly distributed among 10 cell types (shown in fig. 3), with more widely distributed cell types being intestinal epithelial cells of the intestinal tract, adrenal cortex cells of the adrenal gland, ureteric bud cells of the kidney, and acinar cells of the pancreas. In addition, some amniotic fluid cells are derived from microglia of the brain, thymus cells of the thymus, vascular endothelial cells, neutrophils, bronchioles of the lung and epithelial cells of the alveoli, and interstitial cells. While according to the knowledge of the development of the embryo, the intestinal epithelium is mainly derived from the midgut part of the original gut, the adrenal glands and kidneys are derived from the mesoderm, and the pancreas is mainly derived from the foregut part of the original gut, i.e. amniotic fluid cells are mainly derived from cells of the endodermal and mesodermal layers. Some amniotic cells are microglia derived from nervous system, which develop from ectoderm, but have a small cell content.
By the method, 10 cell types are identified in the amniotic fluid in the batch, wherein the cell types with more amniotic fluid cell distribution are intestinal epithelial cells of intestinal tracts, adrenal cortex cells of adrenal glands, ureteral bud cells of kidneys and acinar cells of pancreas. The cells accounting for a large number are mostly derived from mesoderm and endoderm, and cells derived from ectoderm in amniotic fluid samples are microglial cells with less distribution.
The method of the invention carries out cell identity identification aiming at primary cells in amniotic fluid samples, and the cells are not cultured, thereby avoiding 'cell screening' caused by the culture process and avoiding the change of cell types or numbers in the culture process. According to the invention, the fetal cell data in the midgestation stage is referred to for tracing the amniotic fluid cells in the midgestation stage, so that not only is the demonstration that the amniotic fluid cells are derived from the fetus in the midgestation stage, but also the information is sample information in the midgestation stage, and the tracing of the amniotic fluid cells in the midgestation stage is more credible. According to the invention, cell tracing is performed on the amniotic fluid sample, the organ source and the germ layer source of the amniotic fluid cells are described, and cell identity identification is performed on specific cell types through high expression genes, so that the classification and tracing of the cells are more accurate.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (4)
1. A method for identifying the cell type and tracing of primary cells in amniotic fluid samples, comprising the steps of:
s1, capturing single cells in primary cells in a amniotic fluid sample, and sequencing to obtain data of the amniotic fluid sample;
s2, fetal cell data are obtained;
S3, grouping cells and carrying out cluster analysis;
S4, identifying the group without fetal cell distribution through high expression genes;
s5, carrying out identification on amniotic fluid cells and annotating organ sources and germ layers sources;
The S1 primary cells are obtained by the following steps: diluting the amniotic fluid sample by a limiting dilution method, and preparing cells in the amniotic fluid sample into a specific single-cell suspension to obtain single cells; the amniotic fluid sample is derived from a midpregnancy period;
The specific steps of obtaining fetal cell data in S2 are as follows: collecting the gene expression matrix of the cells, the cell type and organ source information of each cell by using loomR bags, and taking 1:100 as a fixed interval, namely taking data information of 1 cell every 100 cells;
The S3 cell grouping specifically comprises the following steps:
1) Respectively constructing Seruat objects from fetal cell data and amniotic fluid sample data, wherein the sample data and the fetal cell data are mapped;
2) Separating cells into different groups (Cluster) by UMAP Cluster analysis;
3) Counting the number of cells of different cell types of fetal data and amniotic fluid sample cells distributed in different groups (Cluster), wherein the most distributed fetal cell type in each group (Cluster) is identified as the cell type of the group (Cluster);
The 3) obtains a plurality of cell types, wherein the identification can be carried out on the group with fetal cell distribution, and the identification of the high expression gene is carried out on the group without fetal cell distribution;
the identification method of the S4 high expression gene specifically comprises the following steps:
1) FINDALLMARKER was used to find highly expressed genes in a cluster without fetal cell distribution;
2) Screening high-expression genes of a group (Cluster) without fetal cell distribution, extracting top50, and setting filtering and screening conditions as follows: avg_ logFC > =2, pct.1> =0.4, and pct.2> =0.4.
3) Cell identification is carried out on the high-expression genes of the group (Cluster) without fetal cell distribution by using CELLMARKER database;
The specific steps of S5 annotating amniotic fluid cells with organ sources and germ layer sources are as follows: based on organ origin, germ layer origin was determined.
2. The method of claim 1, wherein the specific steps of capturing single cells and sequencing in S1 are: single cell capturing is completed through a single cell capturing platform; the mRNA is then captured for reverse transcription and library-building sequencing.
3. The method of claim 1, wherein the gestational age of the fetus is about the week of gestation with the amniotic fluid sample to be analyzed.
4. Use of the method according to any one of claims 1-3 for identifying and tracing cell types of primary cells in amniotic fluid samples, said use being for non-diagnostic and therapeutic purposes.
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