Sorting method of skeleton-derived hematopoietic stem cells applicable across animal categories

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a separation method of skeleton-derived hematopoietic stem cells applicable across animal categories. The method provided by the invention is used for sorting based on the capability of the cells to retain nucleic acid dye, and is particularly suitable for separating invertebrate stem cells lacking specific antibodies. The BSCS living cell nucleic acid dye used in the invention can emit blue fluorescence under the excitation of ultraviolet light, and the stem cells highly express ABC transporter protein, so that compared with other cell types, the BSCS living cell nucleic acid dye has stronger dye excretion capability. Therefore, when the mixed cell suspension dyed for a long time is subjected to flow cytometry, the stem cell population can show lower fluorescence intensity in the V-450 channel. The low toxicity of the BSCS ensures the minimum damage to the cells; the dependence of a traditional method on a specific surface antibody is broken through; the method has wide applicability and can be used for separation and purification of most invertebrate stem cell groups lacking specific markers.

Description

Technical Field

[0001] The present invention belongs to the field of cell biotechnology, and in particular relates to a method for sorting bone-derived hematopoietic stem cells applicable across animal classes. Background Art

[0002] The skeleton of vertebrates belongs to the endoskeleton system, primarily composed of an organic matrix composed of collagen and the mineral hydroxyapatite deposited on it. This bone tissue is widely distributed throughout the body and contains a variety of functional bone cells, such as osteoblasts, osteocytes, and osteoclasts, which can dynamically reshape and self-repair under the control of various hormones and physiological signals. In contrast, the skeletal structure of invertebrates is an exoskeleton, located on the body surface and primarily composed of materials such as calcium carbonate or chitin. Traditionally, its formation process is believed to be primarily an extracellular secretion process, lacking direct cellular participation. Its mineralized structure is often viewed as a static, inanimate shell used for support and defense.

[0003] Although the traditional view is that invertebrate exoskeletons are composed of calcium carbonate deposits and lack cell involvement, we have identified for the first time a biologically active cell population within this mineralized tissue. Based on this discovery, we successfully isolated and systematically characterized these cells from shells, revealing a high proportion of stem cell-like cells. Given the lack of clear maturation markers for these cells, we are further committed to establishing a method for the efficient isolation of these marker-free stem cells to promote in-depth research on the cellular composition and biological functions of invertebrate skeletons.

[0004] Currently, a highly standardized technical system has been established for stem cell research in vertebrates. The isolation, identification, and culture of stem cells are highly dependent on specific antibody labeling techniques. Antibody labeling has become a core tool in vertebrate research: nuclear-labeling antibodies targeting pluripotency transcription factors (such as Oct4, Nanog, and Sox2), as well as membrane-labeling antibodies targeting cell surface markers (such as the SSEA series and CD molecules), together constitute a standardized system for stem cell identification. These antibodies are not only used for immunofluorescence localization and flow cytometry, but are also combined with magnetic cell sorting (MACS) and flow cytometry (FACS) technologies to achieve precise stem cell isolation (purity typically >95%).

[0005] However, when these mature vertebrate stem cell research methods are applied to invertebrate systems, they face fundamental technical obstacles. The primary problem lies in antibody labeling: since invertebrate stem cells lack conserved surface markers, and existing commercial antibodies are mainly designed for vertebrate antigens, cross-reactivity is extremely low, which severely limits their isolation and purification. Secondly, in terms of sorting technology, invertebrate stem cells often exist in complex tissues in atypical morphologies, and traditional FACS sorting methods are inefficient due to problems such as cell size heterogeneity and enzyme sensitivity. Summary of the Invention

[0006] The object of the present invention is to provide a method suitable for isolating stem cells from bones of most marine animals.

[0007] The present invention provides a method for extracting bone-derived hematopoietic stem cells applicable across animal phyla. The method comprises: filtering and centrifuging a suspension of marine organism exoskeleton cells to obtain a single-cell suspension; staining the single-cell suspension, and then performing flow cytometry sorting to obtain stem cells in the cell suspension; the dye used for staining is BSCS (Bone Stem Cell Sorting) dye, the components of which are a mixture of Vybrant DyeCycle Violet and Hoechst.

[0008] Preferably, the filtration comprises the following steps: filtering with a 100 μm cell sieve and then filtering again with a 40 μm cell sieve.

[0009] Preferably, the centrifugal speed is 800g-1000g, the time is 3-5min, and the temperature is 3°C-5°C.

[0010] Preferably, the volume ratio of the Vybrant DyeCycle Violet to Hoechst is 1:1-1.5.

[0011] Preferably, the staining comprises the following steps: after centrifugation and enrichment of the single cell suspension, the supernatant is discarded, and the cells are resuspended with 3×PBS, 1%-2% volume of BSCS (Bone stem cell sorting) dye is added to the suspension after resuspension, and the cells are stained for 1-1.5 hours in the dark to obtain stained cells, and the environmental temperature for the staining is 2°C-6°C.

[0012] Preferably, after the staining is completed, the stained cells are subjected to the following treatments: washing with 3×PBS 2-3 times, and then resuspending with filtered seawater to a cell concentration of 1-10×10 6 cells / mL.

[0013] Preferably, the sheath fluid used in the flow sorting is filtered seawater containing 5-10 mM HEPES; the filtered seawater is seawater that has passed through a 0.1 μm nylon filter membrane.

[0014] Preferably, the flow sorting conditions are as follows: sample flow rate 2000-3000 cells / s, sample mixing speed 400-600 r / min, V-450 and V-660 gains are default values, and cells with low V-450 are selected.

[0015] The method provided by the present invention, which utilizes the ability of cells to retain nucleic acid dyes for sorting, is particularly suitable for isolating invertebrate stem cells that lack specific antibodies. Therefore, the application of the method described in the above technical solution to isolating invertebrate stem cells should also be included in the scope of protection of the present invention.

[0016] Beneficial effects of the present invention: The method provided by the present invention is based on the ability of cells to retain nucleic acid dyes for sorting, and is particularly suitable for the separation of invertebrate stem cells that lack specific antibodies. Its core is the use of BSCS, a live cell nucleic acid dye that can emit blue fluorescence under ultraviolet light excitation. Since stem cells highly express ABC transporters, they have a stronger dye efflux capacity than other cell types. Therefore, when a mixed cell suspension stained for a long time is subjected to flow cytometry analysis, the stem cell population will show a lower fluorescence intensity in the V-450 channel. This feature enables us to efficiently separate stem cell populations. This method has multiple advantages: the low toxicity of BSCS ensures minimal damage to cells; it breaks through the traditional method's reliance on specific surface antibodies; it has wide applicability and can be used for the separation and purification of most invertebrate stem cell populations that lack specific markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 Schematic diagram of flow cytometry for separation of scallop shell cells in Example 1; Figure 2 In accordance with Figure 1 Observation results of conditionally sorted scallop cells; Figure 3 Vertebrate stem cell-related genes Figure 1 Relative expression of sorted cells; Figure 4 This is a schematic diagram of flow cytometry sorting of sea urchin shell cells in Example 2; Figure 5 In accordance with Figure 4Observation results of conditionally sorted sea urchin cells; Figure 6 This is a schematic diagram of flow cytometry sorting of swimming crab shell cells in Example 3; Figure 7 In accordance with Figure 6 Observation results of conditionally sorted swimming crab shell cells; Figure 8 Schematic diagram of flow cytometry sorting of turbot skeletal cells in Example 4; Figure 9 In accordance with Figure 8 Observation results of conditionally sorted turbot skeletal cells; Figure 10 This is the observation result of the scallop shell cells with a staining time of 5 minutes; Figure 11 The results show the observation of scallop shell cells after staining for 4 hours. DETAILED DESCRIPTION

[0019] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0021] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0022] Example 1 Research object: Representative animal of the mollusk group, the scallop (1) After obtaining the cell suspension of the scallop shell, first use a 100 μm cell sieve to filter out large tissue fragments that are not completely dissociated to obtain a cell suspension mixed with smaller tissue fragments.

[0023] (2) Then use a 40 μm cell sieve again to filter out the single cells in the cell suspension to obtain a clean single-cell suspension.

[0024] (3) Add 1%-2% volume of BSCS dye (Vybrant DyeCycleViolet:Hoechst volume ratio = 1:1) to the above single-cell suspension and mix well with a pipette. Stain for 1 hour in the dark. The ambient temperature during staining is 2℃-6℃.

[0025] (4) After staining, centrifuge the cells at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in 3× PBS.

[0026] (5) Centrifuge again at 800 g for 5 min in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in filtered seawater.

[0027] (6) The resuspended cells were loaded onto the flow cytometer for flow cytometry sorting. The sheath fluid used for flow cytometry sorting was sterilized seawater with HEPES (5-10 mM) added. The nozzle used for sorting had a size of 100 μm, the sample flow rate was 2000-3000 cells / s, the sample mixing speed was approximately 500 rpm, and the gains of the V-450 and V-660 were set to the default values.

[0028] (7) During sorting, the X axis is V-660 and the Y axis is V-450. When sorting, select cells with low V-450 and select cells with 10 3 -10 4 In the interval, circle the sorting gate of cluster1. 5 -10 6 Circle the sorting gate for cluster 2. Sort the cells in the two gates into new centrifuge tubes for subsequent experiments.

[0029] Figure 1 This is a schematic diagram of flow cytometry sorting of scallop cells. The results in the figure show that stem cells are concentrated in the 10 3 -10 4 interval, the other non-stem cell populations are concentrated in 10 5 During sorting, cells in the two gates were sorted out and used for downstream experiments.

[0030] Figure 2 In accordance with Figure 1 Microscopic observation of the cells sorted by the gate showed that the stem cells in cluster 1 showed an appearance of a high nuclear-to-cytoplasmic ratio, while the non-stem cells in cluster 2 showed an appearance of a low nuclear-to-cytoplasmic ratio.

[0031] Vertebrate stem cell-related genes Figure 2 The expression of the sorted cells is as follows Figure 3 shown.

[0032] Example 2 Research object: Sea urchin, a representative animal of the echinoderms group (1) After obtaining the cell suspension of sea urchin shells, first use a 100 μm cell sieve to filter out large tissue fragments that are not completely dissociated to obtain a cell suspension mixed with smaller tissue fragments.

[0033] (2) Then use a 40 μm cell sieve again to filter out the single cells in the cell suspension to obtain a clean single-cell suspension.

[0034] (3) Add 1%-2% volume of BSCS dye (Vybrant DyeCycleViolet:Hoechst volume ratio = 1:1) to the above single-cell suspension and mix well with a pipette. Stain for 1 hour in the dark. The ambient temperature during staining is 2℃-6℃.

[0035] (4) After staining, centrifuge the cells at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in 3× PBS.

[0036] (5) Centrifuge again at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in filtered seawater.

[0037] (6) The resuspended cells were loaded onto the flow cytometer for flow cytometry sorting. The sheath fluid used for flow cytometry sorting was sterilized seawater with HEPES (5-10 mM) added. The nozzle used for sorting had a size of 100 μm, the sample flow rate was 2000-3000 cells / s, the sample mixing speed was approximately 500 rpm, and the gains of the V-450 and V-660 were set to the default values.

[0038] (7) During sorting, the X axis is V-660 and the Y axis is V-450. When sorting, select cells with low V-450 and select cells with 10 4 In the upper and lower ranges, circle the sorting gate of P1. 5 Circle the P2 sorting gate in the upper and lower intervals. Sort the cells in the two gates into new centrifuge tubes for subsequent experiments.

[0039] Figure 4 This is a schematic diagram of flow cytometry sorting of sea urchin cells. The results in the figure show that stem cells are concentrated in the 10 4 The other non-stem cell populations are concentrated around 10 5 Near the interval.

[0040] Figure 5 In accordance with Figure 4 Microscopic observation of the cells sorted by the drawn gates showed that the stem cells in P1 had a high nuclear-to-cytoplasmic ratio, while the non-stem cells in P2 had a low nuclear-to-cytoplasmic ratio.

[0041] Example 3 Research object: swimming crab, a representative animal of the arthropod group (1) After obtaining the cell suspension of the swimming crab shell, first use a 100 μm cell sieve to filter out large tissue fragments that are not completely dissociated to obtain a cell suspension mixed with smaller tissue fragments.

[0042] (2) Then use a 40 μm cell sieve again to filter out the single cells in the cell suspension to obtain a clean single-cell suspension.

[0043] (3) Add 1%-2% volume of BSCS dye (Vybrant DyeCycleViolet:Hoechst volume ratio = 1:1) to the above single-cell suspension and mix well with a pipette. Stain for 1 hour in the dark. The ambient temperature during staining is 2℃-6℃.

[0044] (4) After staining, centrifuge the cells at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in 3× PBS.

[0045] (5) Centrifuge again at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in filtered seawater.

[0046] (6) The resuspended cells were loaded onto the flow cytometer for flow cytometry sorting. The sheath fluid used for flow cytometry sorting was sterilized seawater with HEPES (5-10 mM) added. The nozzle used for sorting had a size of 100 μm, the sample flow rate was 2000-3000 cells / s, the sample mixing speed was approximately 500 rpm, and the gains of the V-450 and V-660 were set to the default values.

[0047] (7) During sorting, the X axis is V-660 and the Y axis is V-450. During sorting, cells with low V-450 are selected, and their values ​​are between 10 3 -10 4 The cells in this interval were sorted into new centrifuge tubes for subsequent experiments.

[0048] Figure 6 This is a schematic diagram of flow cytometry sorting of swimming crab cells. The results in the figure show that stem cells are concentrated in the 10 3 -10 4 interval, the other non-stem cell populations are concentrated in 10 4 The above areas.

[0049] Figure 7 In accordance with Figure 6 Microscopic observation of the cells sorted by the drawn gates showed that the stem cells in P1 had a high nuclear-to-cytoplasmic ratio, while the non-stem cells in P2 had a low nuclear-to-cytoplasmic ratio.

[0050] Example 4 Research object: Turbot, a representative vertebrate group (1) After obtaining the cell suspension of turbot bones, first use a 100 μm cell sieve to filter out large tissue fragments that are not completely dissociated to obtain a cell suspension mixed with smaller tissue fragments.

[0051] (2) Then use a 40 μm cell sieve again to filter out the single cells in the cell suspension to obtain a clean single-cell suspension.

[0052] (3) Add 1%-2% volume of BSCS dye (Vybrant DyeCycleViolet:Hoechst volume ratio = 1:1) to the above single-cell suspension and mix well with a pipette. Stain for 1 hour in the dark. The ambient temperature during staining is 2℃-6℃.

[0053] (4) After staining, centrifuge the cells at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in 3× PBS.

[0054] (5) Centrifuge again at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in filtered seawater.

[0055] (6) The resuspended cells were loaded onto the flow cytometer for flow cytometry sorting. The sheath fluid used for flow cytometry sorting was sterilized seawater with HEPES (5-10 mM) added. The nozzle used for sorting had a size of 100 μm, the sample flow rate was 2000-3000 cells / s, the sample mixing speed was approximately 500 rpm, and the gains of the V-450 and V-660 were set to the default values.

[0056] (7) During sorting, the X axis is V-660 and the Y axis is V-450. During sorting, cells with low V-450 are selected, and their values ​​are usually around 10 3 -10 4 The cells in this interval were sorted into new centrifuge tubes for subsequent experiments.

[0057] Figure 8 Schematic diagram of flow cytometry sorting of turbot skeletal cells. The results in the figure show that stem cells are concentrated in the 10 5 The following areas, other non-stem cell populations are concentrated in 10 5 and above areas.

[0058] Figure 9 In accordance with Figure 8 Microscopic observation of the cells sorted by the drawn gates showed that the stem cells in P1 had a high nuclear-to-cytoplasmic ratio, while the non-stem cells in P2 had a low nuclear-to-cytoplasmic ratio.

[0059] Example 5 Research object - the effect of different staining time on sorting results (1) To verify the effect of different staining times on the sorting results when using BSCS dye (Vybrant DyeCycle Violet: Hoechst volume ratio = 1:1) for cell staining, a shorter staining time (5 min) and a longer staining time (4 h) were set, and then compared with the 1 h staining time scheme to optimize the staining time.

[0060] The control sample was a cell suspension of scallop shells. The processing steps were the same as those in Example 1: first, a 100 μm cell sieve was used to filter out incompletely dissociated large tissue fragments to obtain a cell suspension mixed with smaller tissue fragments.

[0061] (2) Then use a 40 μm cell sieve again to filter out the single cells in the cell suspension to obtain a clean single-cell suspension.

[0062] (3) Add 1%-2% volume of BSCS dye to the above single-cell suspension and mix well with a pipette. After staining in the dark for 5 minutes, remove a portion of the cells and proceed with steps (4)-(7). Continue staining the remaining cells in the centrifuge tube for 4 hours, then proceed with steps (4)-(7). The ambient temperature during staining is 2°C-6°C.

[0063] (4) After staining, centrifuge the cells at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in 3× PBS.

[0064] (5) Centrifuge again at 800-1000g for 5 minutes in a pre-cooled centrifuge at 4°C. Discard the supernatant and resuspend the cells in filtered seawater.

[0065] (6) The resuspended cells were loaded onto the flow cytometer for flow cytometry sorting. The sheath fluid used for flow cytometry sorting was sterilized seawater with HEPES (5-10 mM) added. The nozzle used for sorting had a size of 100 μm, the sample flow rate was 2000-3000 cells / s, the sample mixing speed was approximately 500 rpm, and the gains of the V-450 and V-660 were set to the default values.

[0066] (7) During sorting, the X-axis is V-660 and the Y-axis is V-450. Cells with low V-450 are selected during sorting.

[0067] Figure 10 The figure shows the flow cytometry results of 5 min staining time. The results show that the fluorescence signals of most cells are concentrated below 10 3In the region with a 5-min staining, the separation between stem cells and non-stem cells was not obvious. Therefore, effective stem cell separation could not be achieved under the 5-min staining condition.

[0068] Figure 11 The flow cytometry results of the staining time of 4 hours are shown. As can be seen in the figure, the cells are mainly concentrated in the area with a fluorescence intensity of about 10 3 No obvious cell aggregation was observed in other areas. Therefore, effective stem cell sorting could not be achieved under 4-hour staining conditions.

[0069] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for extracting bone-derived hematopoietic stem cells applicable across animal classes, characterized in that: The method comprises: filtering and centrifuging a suspension of marine organism exoskeleton cells to obtain a single cell suspension; staining the single cell suspension, and then performing flow cytometry sorting to obtain stem cells in the cell suspension; The dye used in the dyeing is BSCS dye, and the components of the dye are a mixture of Vybrant DyeCycle Violet and Hoechst.

2. The method according to claim 1, characterized in that The filtration comprises the following steps: filtering with a 100 μm cell sieve and then filtering with a 40 μm cell sieve.

3. The method according to claim 1, characterized in that The centrifugal speed is 800g-1000g, the time is 3-5min, and the temperature is 3°C-5°C.

4. The method according to claim 1, wherein The volume ratio of the Vybrant DyeCycle Violet to Hoechst is 1:1-1.

5.

5. The method according to claim 1, wherein The staining comprises the following steps: after centrifugation and enrichment of a single cell suspension, discarding the supernatant, resuspending the suspension with 3×PBS, adding 1%-2% volume of BSCS dye to the suspension after resuspension, staining in the dark for 1-1.5 hours to obtain stained cells, and the environmental temperature for the staining is 2°C-6°C.

6. The method according to claim 5, characterized in that After the staining was completed, the stained cells were treated as follows: washed 2-3 times with 3×PBS and then resuspended in filtered seawater to a cell concentration of 1-10×10 6 cells / mL; the filtered seawater is seawater that has passed through a 0.1 μm nylon filter membrane.

7. The method according to claim 1, characterized in that The sheath fluid during flow sorting is filtered seawater containing 5-10 mM HEPES; the filtered seawater is seawater that has passed through a 0.1 μm nylon filter membrane.

8. The method according to claim 1, characterized in that The flow sorting conditions are as follows: sample flow rate 2000-3000 cells / s, sample mixing speed 400-600 r / min, V-450 and V-660 gains are set to default values, and cells with low V-450 are selected.

9. Use of the method according to any one of claims 1 to 8 in isolating invertebrate stem cells.

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