CN113122505A - Method for obtaining urine source induced pluripotent stem cells through retrovirus - Google Patents
Method for obtaining urine source induced pluripotent stem cells through retrovirus Download PDFInfo
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Abstract
The invention relates to a method for obtaining urine source induced pluripotent stem cells by retrovirus, which comprises the following steps; step one, urine collection and urine primary cells acquisition; step two, packaging of retrovirus; infection of urine cells; step four, inducing urine cells; fifthly, picking out cloned cells; and step six, identifying the urine source iPSC. The method can remove the complicated and uncontrollable mouse embryo fibroblast preparation, changes the commercialized matrigel into the commercialized matrigel, reduces the process links and the experimental difficulty, and simultaneously adopts a micromolecule two-stage induction method to shorten the induction time.
Description
Technical Field
The invention relates to a method for obtaining urine source induced pluripotent stem cells through retrovirus.
Background
The main steps of obtaining the urine-induced pluripotent stem cells in the prior art (T Z, C B, S D, et al. Generation of human induced pluripotent stem cells from urine samples. Nature protocols.2012; 7(12): 2080-: 1. collecting urine and obtaining primary urine cells; 2. packaging retrovirus and infecting urine primary cells; 3. the infected urine primary cells are transferred to mouse embryonic fibroblasts for continuous induction. Wherein, the preparation of the mouse embryo fibroblast used in the step 3 needs to prepare the trunk of a fetal mouse of a CF-1 mouse which is pregnant for 13.5 days into primary cells and then to be treated by gamma ray irradiation or mitomycin C, the intensity and time length of the gamma ray irradiation or the concentration of the mitomycin C need to be found out in the treatment process, the conditions are difficult to control, the process is complex, and simultaneously, the safety of inducing pluripotent stem cells is reduced by the cells from the mouse as the primer in the induction process. According to the scheme, the 3 rd step of the induction process in the scheme is improved, complicated and uncontrollable mouse embryo fibroblast preparation is removed, the commercialized matrigel is used instead, the process links are reduced, the experiment difficulty is reduced, and meanwhile, the small molecule two-stage induction method is adopted, so that the induction time is shortened.
Disclosure of Invention
The invention provides the following technical scheme:
a method for obtaining urine-derived induced pluripotent stem cells by retrovirus comprises the following steps;
step one, urine collection and urine primary cells acquisition;
step two, packaging of retrovirus;
infection of urine cells;
step four, inducing urine cells;
fifthly, picking out cloned cells;
and step six, identifying the urine source iPSC.
Preferably, the third step comprises preparing a pre-matrigel coating, and the specific steps are as follows: processing and subpackaging matrigel; in use, the dispensed matrigel was removed and mixed with pre-cooled Duchen modified eagle's medium/Hanchen F-12 nutrient medium according to a ratio of 1: 100 are diluted and then evenly spread in a culture dish, 8 ml/dish of a 100mm culture dish, 1 ml/hole of a 6-hole plate and 500 ul/hole of a 12-hole plate; incubate at room temperature for at least 1 hour or 37 ℃ for at least 30 minutes, and aspirate the remaining matrigel dilution before use.
Preferably, step three includes step 3.1: spreading 60000 urine cells in each well of the 6-well plate; observing urine cell confluency of about 30% 24 hours after inoculation, removing culture medium and adding 2ml fresh kidney epithelial cell/interstitial cell amplification culture medium; subsequently, 8ml of the four-element virus supernatant collected in the first step of item 4 of the second step was added to each well; cultured at 37 ℃ in a 95% CO2 environment.
Preferably, step three includes step 3.2: after 12-16 hours, remove the medium and add 2ml of fresh renal epithelial/mesenchymal cells expansion medium per well to allow cell recovery; after 8-12 hours, adding 8ml of four-factor virus supernatant collected in the second 4 strips in the second step into each hole for second infection; cultured at 37 ℃ in a 95% CO2 environment.
Preferably, step three includes step 3.3: 12 hours after secondary infection, remove medium and add 2ml of new renal epithelial cell/interstitial cell expansion medium; the culture was continued, and the fresh kidney epithelial cell/mesenchymal cell expansion medium was changed every day.
Preferably, step three includes step 3.4: infected urine cells were observed under a microscope every day.
Preferably, step three includes step 3.5: when the urine cell morphology is obviously changed and reaches 80-90% confluence degree, digesting by using 0.25% pancreatin, terminating digestion by using 10ml of human renal epithelial cell/interstitial cell amplification culture medium, and centrifuging for 5 minutes at room temperature of 200 g; after discarding the supernatant, resuspending the supernatant by using 4ml of renal epithelial cell/interstitial cell amplification culture medium, counting, paving 10 ten thousand infected urine cells in each hole of a 6-hole plate, and adding the human renal epithelial cell/interstitial cell amplification culture medium to 2ml, which is the 1 st day of culture;
preferably, in the step one, the urine collection and the urine primary cell acquisition include the following steps:
step 1.1: collecting urine; step 1.2, separating urine; step 1.3 urine cell amplification;
preferably, in step two, the packaging of the retrovirus comprises the following steps:
step 2.1: according to the following steps: 3 passage laying 5-hole human embryonic kidney 293T cells of 6-hole plate;
step 2.2: preparing infection compound, and packaging vector pCL-Ampho, 4ug gene expression vector (pMXs-OCT3/4, pMXs-KLF4, pMXs-SOX2, pMXs-cMyc or pMXs-GFP1), 24ul polyethyleneimine according to 200ul of reduced serum culture medium per well; after mixing, the mixture was left to stand for 15 minutes.
Step 2.3: during the standing period, the culture medium of the human embryonic kidney 293T cells is replaced by a Du's modified eagle's culture medium preheated to 37 ℃ in advance; after 15 minutes, the mixture is added dropwise and then is shaken evenly; culturing at 37 deg.C in 95% CO2 environment; after 4h, the medium was replaced with pre-warmed serum.
Step 2.4: pMXs-GFP holes are observed after 24 hours of transfection, and the transfection efficiency is ensured to be close to 100%; the supernatant from another 4 wells was collected and 2ml of pre-warmed human embryonic kidney 293T cell culture medium was added again; the collected supernatant was filtered through a 0.22 μm filter, and polybrene was added to a final concentration of 8ug/ml for the first infection of urine cells; after 24 hours, the supernatant was collected for a second time, filtered through a 0.22 micron filter and polybrene was added to a final concentration of 8ug/ml for a second infection of urine cells.
Preferably, in step four, packaging of the retrovirus comprises the steps of:
step 4.1: inducing infected urine cells by adopting a two-stage induction method, and adding five small molecules including A-83-01, CHIR99021, thiavivin, cyclic pifithrin-alpha and sodium butyrate (ACTPN) into a renal epithelial cell/interstitial cell amplification culture medium in the first stage; second stage at mTeSRTM1 culture medium (STEMCELL technologies) containing five kinds of ACTPN and PD0325901 (ACTPNPD for short)2；
Step 4.2: culturing on day 2 without changing culture medium, culturing on day 3 to day 10 with kidney epithelial cell/interstitial cell amplification culture medium + ACTPN, and changing culture medium every other day; culturing at 11-18 days with mTeSRTM1+ ACTPNPD, changing liquid every other day; in the culture process, cell clusters growing like clones gradually appear, the arrangement is compact, the boundary is clear, and the nuclear-cytoplasmic ratio is increased;
step 4.3: starting on day 19 of culture with mTeSRTM1, culturing in a culture medium, and changing the culture solution every day.
Compared with the prior art, the invention has the beneficial effects that:
the scheme used in the invention improves the 3 rd step of the induction process in the prior art, removes the complicated and uncontrollable mouse embryo fibroblast preparation, changes the commercialized matrigel into the traditional Chinese medicine, reduces the process links and the experimental difficulty, and simultaneously adopts a micromolecule two-stage induction method to shorten the induction time.
Description of the drawings:
FIG. 1 is a schematic representation of when urine primary cells are passable;
FIG. 2 is a schematic diagram of the morphology of urine cells on the third day (4-fold mirror) after infection at step 3.4;
FIG. 3 is a schematic representation of the morphology of urine cells at the fourth day (4-fold mirror) after infection at step 3.4;
FIG. 4 is a schematic representation of the morphology of urine cells at the fourth day (10-fold mirror) after infection at step 3.4;
FIG. 5 is a schematic representation of the morphology of urine cells at the fourth day after infection (20-fold mirror) in step 3.4;
FIG. 6 is a schematic diagram of the urine cell morphology of the control group (4-fold mirror) on day four without infecting urine primary cells at step 3.4;
FIG. 7 is a graphical representation of the morphology of urine cells with a confluency of about 90% at day 6 post-infection at day six post-infection at step 3.5;
FIG. 8 is a schematic representation of the cell mass with clonotype growth visible on day seven of the two-stage induction in step 4.2;
FIG. 9 is a schematic representation of the cell pellet becoming more compact on day nine of the two-stage induction in step 4.2;
FIG. 10 is a schematic representation of a clone partially reprogrammed with the prior art technique (day 7 after emergence) at step 4.3;
FIG. 11 is a schematic representation of a fully reprogrammed clone appearing at day 19 of culture using the prior art in step 4.3;
FIG. 12 is a schematic representation of prior art culture of clones of FIG. 11 at day 26 of culture in step 4.3;
FIG. 13 is a schematic representation of day 2 after picking out the clone of FIG. 12 in step 4.3 using prior art culture;
FIG. 14 is a schematic representation of day 6 after colony picking in prior art FIG. 12 in step 4.3;
FIG. 15 is a schematic representation of a fully reprogrammed clone at day 6 (4-fold mirror) grown using the inventive protocol at step 4.3;
FIG. 16 is a schematic view of the clone in FIG. 15 at day 7 (10 fold mirror) after it was picked up in step 4.3 using the technique of the present invention;
FIG. 17 shows the identification of cloned human embryonic stem cell surface markers obtained in step 4.3 using the protocol of the present invention;
FIG. 18 is the identification of the cloned human embryonic stem cell pluripotent gene obtained by the technical solution of the present invention in step 4.3;
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments.
Thus, the following detailed description of the embodiments of the invention is not intended to limit the scope of the invention as claimed, but is merely representative of some embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments of the present invention and the features and technical solutions thereof may be combined with each other without conflict.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
A method for obtaining urine-derived induced pluripotent stem cells by retrovirus comprises the following steps;
step one, urine collection and urine primary cells acquisition;
step two, packaging of retrovirus;
infection of urine cells;
step four, inducing urine cells;
fifthly, picking out and culturing the cloned cells;
and step six, identifying the urine source iPSC.
The first embodiment is as follows:
in the first step, the urine collection and the acquisition of urine primary cells comprise the following steps:
step 1.1 urine collection: urine was collected in a sterile container of appropriate volume. Before collection, donors are allowed to drink as much water as possible, and if the collection cannot be immediately processed, 1ml of penicillin/streptomycin solution can be added into a sterile container in advance in a biological safety cabinet to prevent bacterial contamination. Before collection, the urethra area is wiped by wet tissues, the front section urine is discarded, the middle section urine is collected, and the inner wall of the container is prevented from being polluted as much as possible in the collection process. Increasing the amount of urine increases yield. The collected material needs to be treated as soon as possible, and if the collected material cannot be treated immediately, the collected material needs to be stored at 4 ℃ and treated within 4 hours.
Step 1.2 urine separation: all operations, starting from the urine isolation step, should be performed in an environment compatible with cell culture to avoid contamination. Transfer the urine to a 50ml centrifuge tube, e.g., multiple centrifuge tubes may be used for more urine. Centrifuge at 400g for 10 minutes at room temperature, carefully discard the supernatant leaving about 1ml in the tube, resuspend and transfer to a new 50ml centrifuge tube, if multiple tubes are used in the same centrifuge tube. 10ml of wash buffer was added, centrifuged at 200g for 10 min at room temperature, the supernatant was carefully discarded, and approximately 0.2ml of cell pellet remained in the tube. 1ml of initial medium was added to resuspend the cell pellet and the cell suspension was transferred to 1 well of a 12-well plate (pre-coated with 0.1% gelatin). 1ml of the initial medium was added thereto, and the mixture was incubated at 37 ℃ for 24 hours.
Step 1.3 urine cell expansion:
preparing materials:
washing buffer solution: the washing buffer was Du's phosphate buffer plus 100ug/ml primary cell antibiotic (primocin, InvivoGen, cat. No. ant-pm-1). 500ml of buffer, 1ml of primary cell antibiotic, and 499ml of Duchen phosphate buffer were prepared. (stored at 4 ℃ and used up within 2 weeks)
0.1% gelatin coated petri dish: an appropriate volume of 0.1% gelatin was added to the dish to ensure liquid coverage of the entire dish bottom surface and was used after incubation at 37 ℃ for about 30 minutes. The gelatin is sucked away before use and is washed once by phosphate buffer solution for use.
Initial medium: the initial medium was Du's modified eagle's medium/Han's F-12 nutrient medium plus 10% fetal bovine serum, 100ug/ml primary cell antibiotic and renal epithelial cell growth medium supplement (Lonza, cat. No. CC-4127). 500ml is prepared, 50ml of fetal bovine serum and 1ml of primary cell antibiotics are added, 1 set of renal epithelial cell growth medium is filled with additives, and the medium is supplemented to 500ml by Du's modified eagle's medium/Han's F-12 nutrient medium. (the additive tubes were carefully washed with the initial medium to avoid loss) (stored at 4 ℃ in the dark, used within 2 weeks).
Kidney epithelial cell expansion medium: 500ml of kidney epithelial cell amplification culture medium is prepared, and the whole set of kidney epithelial cell growth culture medium additive is added into 500ml of kidney epithelial cell basic culture medium (Lonza, cat.no. CC-3190).
Interstitial cell amplification medium: the interstitial cell amplification culture medium is a high-sugar Du's modified eagle culture medium added with 10% fetal calf serum, 1% glutamine, 1% non-essential amino acid solution, 100U/ml penicillin, 100ug/ml streptomycin, 5ng/ml recombinant human fibroblast growth factor, 5ng/ml recombinant human platelet-derived growth factor-AB and 5ng/ml recombinant human epidermal growth factor. 500ml of interstitial cell amplification culture medium, 50ml of fetal calf serum, 5ml of glutamine, 5ml of nonessential amino acid solution, 2.5ml of penicillin/streptomycin solution, 5ng/ml of recombinant human fibroblast growth factor, 5ng/ml of recombinant human platelet-derived growth factor-AB and 5ng/ml of recombinant human epidermal growth factor are prepared. After filtration through a 0.22 micron filter, 500ml was supplemented with high-sugar Duchen modified eagle's medium.
Kidney epithelial cell/stromal cell expansion medium: the kidney epithelial cell/interstitial cell amplification culture medium is prepared by mixing a kidney epithelial cell amplification culture medium and an interstitial cell amplification culture medium in a ratio of 1:1, preparing 500ml of the kidney epithelial cell/interstitial cell amplification culture medium, and mixing 250 ml of the kidney epithelial cell amplification culture medium and 250 ml of the interstitial cell amplification culture medium (stored at 4 ℃ in a dark place and used within 2 weeks).
The specific implementation process comprises the following steps: 1ml of the initial medium was added 24 hours, 48 hours, and 72 hours after plating. Do not remove any media. After 96 hours of plating, most of the medium was removed, 1ml was left, and 1ml of renal epithelial/stromal cell expansion medium was added. Medium for renal epithelial cell/mesenchymal cell expansion was changed half a day. When the cells can be passaged as shown in FIG. 1 (80-90% confluency after 9-12 d), the digestion of the cells to 12 well plates continued to expand 1 new well (about 100000 cells, if the cell density was high, to 6 well plates), which is passage 1. (cells were able to tolerate 0.25% pancreatin, stopped without serum-containing medium, and used pancreatin inhibitors). And (3) changing the liquid every other day, and when the cell reaches 80-90% confluence degree, performing the steps of: 4 digestion of the passaged cells to appropriate culture dishes for further expansion, or plating onto 6-well plates for infection. This is generation 2. (the urine cells which can achieve the highest efficiency of inducing the generation of the pluripotent stem cells are from generation 1 to generation 3) are subsequently used, and the cells are frozen by using cell freezing solution without serum and can be frozen at the temperature of minus 80 ℃ or liquid nitrogen.
Example two:
in the second step, packaging of the retrovirus comprises the following steps:
preparing materials: human embryonic kidney 293T cell culture medium: du's modified eagle's medium plus 10% fetal bovine serum.
The specific implementation process comprises the following steps:
step 2.1: the human embryonic kidney 293T cells with good growth condition are used for packaging retrovirus, namely the growth rate is 1: the confluence rate of 80% can be achieved after 3 passages. According to the following steps: human embryonic kidney 293T cells plated in 5 wells of 6-well plates at 3 passages showed a cell confluence of 80% after 24 hours (no antibiotic added in culture, otherwise the transfection efficiency was reduced).
Step 2.2: the infection complex was prepared by 200ul of reduced serum medium per well, 4ug of packaging vector pCL-Ampho, 4ug of gene expression vector (pMXs-OCT3/4, pMXs-KLF4, pMXs-SOX2, pMXs-cMyc or pMXs-GFP1), 24ul of polyethyleneimine. After mixing, the mixture was left to stand for 15 minutes.
Step 2.3: during the standing period, the human embryonic kidney 293T cell culture medium was replaced with Du's modified eagle's medium pre-warmed to 37 ℃. After 15 minutes the mixture was added dropwise and shaken gently. Culturing at 37 deg.C in 95% CO2 environment. After 4h, the medium was replaced with pre-warmed serum.
Step 2.4: pMXs-GFP wells were observed 24 hours after transfection to ensure near 100% transfection efficiency. Supernatants from 4 additional wells were collected and 2ml of pre-warmed human embryonic kidney 293T cell culture medium was added again. The collected supernatant was filtered through a 0.22 μm filter, and then polybrene was added to make a final concentration of 8ug/ml for the first infection of urine cells. After 24 hours, the supernatant was collected for a second time, filtered through a 0.22 micron filter and polybrene was added to a final concentration of 8ug/ml for a second infection of urine cells.
Example three:
in step three, the infection of urine cells comprises the following steps:
preparing materials: pre-matrigel coating: by using
hESC-qualified Matrix (cat # 354277). Handling and dispensing of matrigel according to the instructions, especially taking care that the articles in contact with matrigel need to be pre-cooled beforehand, since matrigel starts to set at temperatures above 10 ℃ and cannot be used after setting. In use, the dispensed matrigel was removed and mixed with pre-cooled Duchen modified eagle's medium/Hanchen F-12 nutrient medium according to a ratio of 1: 100 diluted solution is evenly spread in a culture dish, 8 ml/dish of a 100mm culture dish, 1 ml/well of a 6-well plate and 500 ul/well of a 12-well plate. Incubate at room temperature for at least 1 hour or 37 ℃ for at least 30 minutes, and aspirate the remaining matrigel dilution before use.
The specific implementation process comprises the following steps:
step 3.1: 60000 urine cells (best passage 1 to passage 3) were plated in each well of a 6-well plate (0.1% gelatin coating). Urine cells were observed to confluence at about 30% 24 hours after inoculation, and medium was removed plus 2ml of fresh renal epithelial/stromal cell expansion medium. A total of 8ml of the four-element viral supernatant collected first in item 4 of the second step was then added per well. Cultured at 37 ℃ in a 95% CO2 environment.
Step 3.2: after 12-16 hours, the medium was removed and 2ml of fresh renal epithelial/mesenchymal cell expansion medium was added per well to allow cell recovery. After 8-12 hours, a total of 8ml of four-element virus supernatant collected in the second 4 th item of the second step was added to each well for a second infection. Cultured at 37 ℃ in a 95% CO2 environment.
Step 3.3: 12 hours after secondary infection, medium was removed and 2ml of fresh renal epithelial/mesenchymal cell expansion medium was added. The culture was continued, and the fresh kidney epithelial cell/mesenchymal cell expansion medium was changed every day.
Step 3.4: infected urine cells were observed under a microscope every day. The urine cell morphology after four-factor infection is obviously changed (as shown in figures 2-6, the cells become smaller and more compact, and the amplification capacity is obviously enhanced). If the morphological changes of the cells are not apparent after infection, probably due to aging after infection of the cells, the probability of using early passage urine cells is reduced)
The cell proliferation capacity was significantly enhanced as seen by comparison from day 3 to day 4 after infection. On day 4 post-infection, changes in cell morphology, including smaller cell shortening and tighter cell arrangement, were seen after infection compared to the control.
Step 3.5: when the urine cell morphology changes obviously and reaches 80-90% confluence, 0.25% pancreatin is used for digestion, 10ml of human renal epithelial cell/interstitial cell amplification culture medium is used for stopping digestion, and then the cells are centrifuged for 5 minutes at room temperature of 200 g. After discarding the supernatant, the cells were resuspended in 4ml of kidney epithelial cell/mesenchymal cell expansion medium and counted, 10 ten thousand infected urine cells were plated per well (pre-matrigel coated) of a 6-well plate, and human kidney epithelial cell/mesenchymal cell expansion medium was added to 2ml, which is day 1 of culture. The liquid change is carried out every other day after the induction process. The culture medium is preheated at 37 ℃ in advance in the induction process and the liquid change in the subsequent induced pluripotent stem cell culture process.
It should be noted that: in the prior art, the treated mouse embryo fibroblast needs to be plated 1-2 days before urine cells are digested. The method comprises the steps of taking the trunk of a CF-1 female mouse pregnant for 13.5 days, cutting, digesting with pancreatin, culturing to obtain primary cells, and treating with mitomycin C when the primary cells grow to the third generation, wherein the concentration and the treatment time of the mitomycin are optimized, so that the treated cells lose the division and proliferation capacity, but can secrete various cell factors including mitogenic factors and cell differentiation inhibiting factors. In addition to mitomycin C, gamma irradiation can be used to treat mouse embryonic fibroblasts, but additional equipment, i.e., a radiation apparatus, is required, and conditions for the intensity and time of radiation are optimized. The difficulties in the whole preparation process include: 1. the time for the female CF-1 mouse to be pregnant needs to be accurately judged; 2. the optimal treatment conditions of mitomycin C or gamma rays are required to be found according to the laboratory conditions; 3. because the treated mouse embryonic fibroblasts do not have the proliferation capacity, new mouse embryonic fibroblasts need to be continuously prepared. In addition to the complexity of the preparation process, the induction of human induced pluripotent stem cells is carried out by adopting mouse embryonic fibroblasts from allogeneic sources, and the problem of cross contamination exists. Therefore, in this step, the commercial matrigel is used for replacement, which not only simplifies the experimental steps, but also avoids the pollution of the induced pluripotent stem cells by the murine cells.
Example four:
in the fourth step, the infection of urine cells comprises the following specific implementation steps:
step 4.1: inducing infected urine cells by adopting a two-stage induction method, and adding five small molecules including A-83-01, CHIR99021, thiavivin, cyclic pifithrin-alpha and sodium butyrate (ACTPN) into a renal epithelial cell/interstitial cell amplification culture medium in the first stage; second stage at mTeSRTM1 culture medium (STEMCELL technologies) containing five kinds of ACTPN and PD0325901 (ACTPNPD for short)2。
Step 4.2: culturing on day 2 without changing culture medium, culturing on day 3 to day 10 with kidney epithelial cell/interstitial cell amplification culture medium + ACTPN, and changing culture medium every other day; the culture was carried out on days 11 to 18 using mTeRTM 1+ ACTPNPD, and the medium was changed every other day. During the culture process, cell clusters growing like clones gradually appear, the arrangement is compact, the boundary is clear, and the nucleus-cytoplasm ratio is increased.
Step 4.3: on day 19, the culture was started in mTeSRTM1 medium and the medium was changed every day.
It should be noted that: in the prior art, the first step of cultivationCulturing with human embryonic stem cell/fetal calf serum culture medium supplemented with sodium valproate (final concentration of 1mM) from 3 days, changing the culture medium every day, culturing with human embryonic stem cell/fetal calf serum culture medium from 10 days, changing the culture medium every day, and culturing with mTeSR from 17 daysTM1 culture medium. However, many partially reprogrammed clones appeared during the culture process (see fig. 10), which had dense bulges in the middle, irregular edges, and often appeared earlier, and appeared at 5 days of the culture process, and then gradually grown up. The true induced pluripotent stem cell clones appeared later and only started to appear after day 17 in the culture (see FIGS. 11-14). The clone is compact and the edge is neat. However, in one induction, 10 ten thousand infected urine cells are planted in total, and partial reprogramming clones appear earlier and much more, and are selected to be 24 in total, and real induced pluripotent stem cells appear later and appear less, and are selected to be 2 in total.
In the method provided by the invention, 10 ten thousand infected urine cells are planted in one induction, partial reprogramming clones are hardly seen, the true clones begin to appear early, cloning can begin to be selected at the 6 th day of culture (see figures 15-16), and a plurality of clones are selected, wherein 12 clones are selected in total. Therefore, the technical scheme provided by the invention has higher efficiency and saves more time.
Example five:
in the fifth step, the cloning of the cells comprises the following specific implementation steps:
in the fourth step of culture, when the mass growing in the clone sample has the characteristics of tight arrangement, clear boundary, increased cell nucleus-to-cytoplasm ratio and the like, and the area of the mass is more than 1 cm by 1 cm, the clone can be picked out (when the clone is too small, the picked clone can cause clone differentiation). The clones can be picked out under microscope by a sterilized gun head into a 12-hole plate pre-paved with matrigel, placed in a cell culture box for 48h to avoid disturbance, and then changed into mTeSR every dayTM1, culture medium. Clones can be passaged after 7 days, and mechanical passaging into new, pre-matrigel-plated 12-well plates is recommended. After 7 days, passage, cryopreservation and identification can be carried out according to a conventional induced pluripotent stem cell culture method. In the morningDuring the culture process of the induced pluripotent stem cells of the next generation, cell differentiation is easy to occur, and the differentiated cells can be scratched and then subjected to passage.
Example six:
in the sixth step, the urine-derived iPSC expresses surface markers of human embryonic stem cells, including SSEA4, NANOG, TRA1-60 and TRA1-81 (see FIG. 17). The urine-derived ipsc (uipsc) expresses pluripotent genes expressed by human embryonic stem cells (hES), including OCT4, SOX2, KLF4, MYC, and urine cells (urine) as controls (see fig. 18).
The above embodiments are only used for illustrating the invention and not for limiting the technical solutions described in the invention, and although the present invention has been described in detail in the present specification with reference to the above embodiments, the present invention is not limited to the above embodiments, and therefore, any modification or equivalent replacement of the present invention is made; all such modifications and variations are intended to be included herein within the scope of this disclosure and the appended claims.
Claims (15)
1. A method for obtaining urine-derived induced pluripotent stem cells by retrovirus, comprising: comprises the following steps;
step one, urine collection and urine primary cells acquisition;
step two, packaging of retrovirus;
infection of urine cells;
step four, inducing urine cells;
fifthly, picking out cloned cells;
and step six, identifying the urine source iPSC.
2. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 1, wherein the third step comprises pre-preparing a matrigel coating, and comprises the following specific steps: processing and subpackaging matrigel; in use, the dispensed matrigel was removed and mixed with pre-cooled Duchen modified eagle's medium/Hanchen F-12 nutrient medium according to a ratio of 1: 100 are diluted and then evenly spread in a culture dish, 8 ml/dish of a 100mm culture dish, 1 ml/hole of a 6-hole plate and 500 ul/hole of a 12-hole plate; incubate at room temperature for at least 1 hour or 37 ℃ for at least 30 minutes, and aspirate the remaining matrigel dilution before use.
3. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 2, wherein the step three comprises the step 3.1: spreading 60000 urine cells in each well of the 6-well plate; observing urine cell confluency of about 30% 24 hours after inoculation, removing culture medium and adding 2ml fresh kidney epithelial cell/interstitial cell amplification culture medium; subsequently, 8ml of the four-element virus supernatant collected in the first step of item 4 of the second step was added to each well; cultured at 37 ℃ in a 95% CO2 environment.
4. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 3, wherein the step three comprises the steps of 3.2: after 12-16 hours, remove the medium and add 2ml of fresh renal epithelial/mesenchymal cells expansion medium per well to allow cell recovery; after 8-12 hours, adding 8ml of four-factor virus supernatant collected in the second 4 strips in the second step into each hole for second infection; cultured at 37 ℃ in a 95% CO2 environment.
5. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 4, wherein the step three comprises the steps of 3.3: 12 hours after secondary infection, remove medium and add 2ml of new renal epithelial cell/interstitial cell expansion medium; the culture was continued, and the fresh kidney epithelial cell/mesenchymal cell expansion medium was changed every day.
6. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 5, wherein the step three comprises the steps of 3.4: infected urine cells were observed under a microscope every day.
7. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 6, wherein the step three comprises the steps of 3.5: when the urine cell morphology is obviously changed and reaches 80-90% confluence degree, digesting by using 0.25% pancreatin, terminating digestion by using 10ml of human renal epithelial cell/interstitial cell amplification culture medium, and centrifuging for 5 minutes at room temperature of 200 g; after discarding the supernatant, the cells were resuspended in 4ml of kidney epithelial cell/mesenchymal cell amplification medium and counted, 10 ten thousand infected urine cells were plated per well of 6-well plate, and human kidney epithelial cell/mesenchymal cell amplification medium was added to 2ml, which is day 1 of culture.
8. The method for obtaining urine-derived induced pluripotent stem cells by retrovirus according to claim 1, wherein the collecting of urine and the obtaining of urine primary cells in the first step comprises the following steps:
step 1.1: collecting urine; step 1.2, separating urine; step 1.3 urine cell expansion.
9. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 1, wherein the packaging of retrovirus in the second step comprises the steps of:
step 2.1: according to the following steps: human embryonic kidney 293T cells plated in 5 wells of 6-well plates were passaged 3.
10. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 9, wherein the packaging of retrovirus in the second step comprises the steps of:
step 2.2: preparing infection compound, and packaging vector pCL-Ampho, 4ug gene expression vector (pMXs-OCT3/4, pMXs-KLF4, pMXs-SOX2, pMXs-cMyc or pMXs-GFP1), 24ul polyethyleneimine according to 200ul of reduced serum culture medium per well; after mixing, the mixture was left to stand for 15 minutes.
11. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 10, wherein the packaging of retrovirus in the second step comprises the steps of:
step 2.3: during the standing period, the culture medium of the human embryonic kidney 293T cells is replaced by a Du's modified eagle's culture medium preheated to 37 ℃ in advance; after 15 minutes, the mixture is added dropwise and then is shaken evenly; culturing at 37 deg.C in 95% CO2 environment; after 4h, the medium was replaced with pre-warmed serum.
12. The method for obtaining urinary derived induced pluripotent stem cells by retrovirus according to claim 11, wherein the packaging of retrovirus in the second step comprises the steps of:
step 2.4: pMXs-GFP holes are observed after 24 hours of transfection, and the transfection efficiency is ensured to be close to 100%; the supernatant from another 4 wells was collected and 2ml of pre-warmed human embryonic kidney 293T cell culture medium was added again; the collected supernatant was filtered through a 0.22 μm filter, and polybrene was added to a final concentration of 8ug/ml for the first infection of urine cells; after 24 hours, the supernatant was collected for a second time, filtered through a 0.22 micron filter and polybrene was added to a final concentration of 8ug/ml for a second infection of urine cells.
13. The method for obtaining urinary induced pluripotent stem cells through retrovirus according to claim 1, wherein the step four comprises the following steps:
step 4.1: inducing infected urine cells by adopting a two-stage induction method, and adding five small molecules including A-83-01, CHIR99021, thiavivin, cyclic pifithrin-alpha and sodium butyrate (ACTPN) into a renal epithelial cell/interstitial cell amplification culture medium in the first stage; second stage at mTeSRTM1 culture medium (STEMCELL technologies) containing five kinds of ACTPN and PD0325901 (ACTPNPD for short)2。
14. The method for obtaining urinary induced pluripotent stem cells by retrovirus according to claim 13, wherein the step four comprises the following steps:
step 4.2: culturing on day 2 without changing culture medium, culturing on day 3 to day 10 with kidney epithelial cell/interstitial cell amplification culture medium + ACTPN, and changing culture medium every other day; culturing for 11 th to 18 th days by adopting mTeRTM 1+ ACTPNPD, and changing the liquid every other day; during the culture process, cell clusters growing like clones gradually appear, the arrangement is compact, the boundary is clear, and the nucleus-cytoplasm ratio is increased.
15. The method for obtaining urinary induced pluripotent stem cells by retrovirus according to claim 14, wherein the step four comprises the following steps:
step 4.3: on day 19, the culture was started in mTeSRTM1 medium and the medium was changed every day.
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| Publication number | Priority date | Publication date | Assignee | Title |
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