Human Endometrial Stem Cell Isolation from Endometrium and Menstrual Blood
从子宫内膜和经血中分离人子宫内膜干细胞   

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Molecular Neurobiology
Oct 2016

 

Abstract

Human endometrial stem cell/stromal cells (hEnSCs) are isolated from endometrium or menstrual blood and are recognized as a valuable cell type in tissue engineering and cell therapy. Furthermore, hEnSCs, which have CD90 (a mesenchymal marker), CD105 (endoglin), CD44, CD146 (endometrial stem cell markers) and lack CD31 (Endothelial marker), CD34 (hematopoietic marker) and CD133 on the cell surface, are a new source of mesenchymal stem/stromal cells. Additionally, these cells can be encapsulated into self-assembling peptide nanofibers as a 3D scaffold for applications in the treatment of neurodegenerative diseases. Here, we describe a protocol to isolate hEnSCs from endometrium or menstrual blood.

Keywords: Endometrial stem cell (子宫内膜干细胞), Endometrial stromal cell (子宫内膜间质细胞), Menstrual blood (经血), Cell isolation (细胞分离), Neural differentiation (神经分化), Self-assembling peptide nanofiber (自组装肽纳米纤维), 3D cell culture (3D细胞培养)

Background

Cell replacement therapy is being studied as a new strategy to manage neurodegenerative diseases such as Alzheimer’ s disease, Stroke, and spinal cord injury (Tavakol et al., 2014b; 2015; 2016a and 2016c). Human endometrial stem cell/stromal cells (hEnSCs) can be isolated from menstrual blood (Azedi et al., 2014 and 2017) and endometrium. These cells have adipogenic (Khanmohammadi et al., 2014), osteogenic (Darzi et al., 2012), and chondrogenic (Kazemnejad et al., 2012) differentiation potential. Furthermore, these cells may be used in combination with scaffolds as an ingredient of smart cell-scaffolds in tissue engineering applications (Tavakol et al., 2014a; 2016b and 2017). It is worth noting that cell compartments such as the exosome, the microvesicle and other components are considered safer than therapies that use the whole cell. hEnSCs are attractive candidates for cell therapy because they are immunosuppressive and have high clonogenicity (1.25%) potential. They also have advantages over other cell types. hEnSCs are better suited for cell therapy than embryonic stem cells because hESCs do not develop teratomas. Unlike mesenchymal stem cells, hEnSCs do not decrease proliferative potency in elderly people (Ebrahimi-Barough et al., 2013). Furthermore, another advantageous feature of hEnSCs is that they can be encapsulated into self-assembling peptide nanofibers and differentiated into neuronal cells (Tavakol et al., 2014c; 2016a; 2016c and 2017). This protocol describes a simple strategy to obtain higher yields of hEnSC from both menstrual blood and endometrium for in-vitro and in-vivo which can be used in numerous studies.

Materials and Reagents

  1. Pipette tips: crystalline, yellow and blue (Gilson, catalog numbers: JHA004 , JHA005 and JHA007 )
  2. SPL cell culture dish, 90 x 15 mm (SPL Life Sciences, catalog number: 11090 )
  3. Falcon tubes 15 ml (SPL Life Sciences, catalog number: 50115 )
  4. PluriStrainer® 70 µm (Pluriselect, catalog number: 43-50070 )
  5. Falcon cell strainer 40 µm (Corning, Falcon®, catalog number: 352340 )
  6. T25 culture flasks (SPL Life Sciences, catalog number: 70125 )
  7. T75 culture flasks (SPL Life Sciences, catalog number: 70175 )
  8. Diva cups (Diva International, Lunette, Finland)
  9. Falcon tubes 50 ml (SPL Life Sciences, catalog number: 50050 )
  10. Endometrium
  11. Menstrual blood
  12. Ficoll-Hypaque (GE Healthcare, Amersham, catalog number: 17-5442-02 )
  13. FITC-conjugated anti CD105 (Abcam, catalog number: ab18278 )
  14. Allophycocyanin-conjugated anti-CD44 antibody (Abcam, catalog number: ab81424 )
  15. Anti-CD34 antibody [EP373Y] (Abcam, catalog number: ab81289 )
  16. Anti-CD133 antibody (Abcam, catalog number: ab19898 )
  17. Penicillin/Streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  18. Amphotrypsin B solution (Sigma-Aldrich, catalog number: A2942 )
  19. Collagenase I (Thermo Fisher Scientific, GibcoTM, catalog number: 17100017 )
  20. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: 806552 )
  21. DMEM-F12 (Thermo Fisher Scientific, GibcoTM, catalog number: 11330057 )
  22. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  23. Trypsin-EDTA 0.25% (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  24. Glutaraldehyde, 25% Aqueous Solution (Merck, catalog number: 354400 )
  25. Ethanol (Merck, catalog number: 818760 )
  26. Fungizone (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  27. Ethylenediaminetetraacetic acid tetrasodium salt dehydrate (EDTA) (Sigma-Aldrich, catalog number: E6511 )
  28. Non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140035 )
  29. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  30. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
  31. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
  32. Isolation media for endometrium (see Recipes)
  33. Plating media (see Recipes)
  34. Basal media for the rest of passage except first passage (see Recipes)
  35. Isolation media for menstrual blood (see Recipes)
  36. Collagenase I (see Recipes)

Equipment

  1. Scalpel handle with scalpel (Fine Science Tools, catalog numbers: 10011-00 , 10003-12 )
  2. Standard sterile forceps (BYDAND, catalog number: BSU101 )
  3. Sterile pipette (SPL LIFE SICENCES, catalog number: 91005 )
  4. 37 °C, 5% CO2 cell culture incubator (New Brunswic Scientific, model: CO-150 )
  5. Centrifuge (Eppendorf, model: 5810 R )
  6. Inverted microscope (Olympus, model: IX51 )
  7. Refrigerator (Pars, model: 1300 )

Software

  1. FlowJo 7.6.1 software (FlowJo, LLC; https://www.flowjo.com)

Procedure

Note: Biopsy of the endometrium must be performed by an expert surgeon in accordance with local ethics policies and guidelines.

  1. To isolate hEnSCs from the endometrium, the following steps are performed
    1. Endometrium specimen biopsy
      The biopsy should be taken from:
      1. Healthy females
      2. Age: between 20-35 years of age
      3. Time: Endometrium biopsy and menstrual blood are isolated on menstrual cycle days 19-24 and 2-4, respectively.
      The biopsy should not be taken from:
      1. Subjects with non-endometrial benign pathological conditions such as polyps, hyperplasia, or cancer.
      2. Subjects that have taken exogenous hormones such as GnRH, progesterone and other hormones for several months prior to the date of the biopsy.
      3. Subjects with endometriosis or any pathological condition in the uterus.
      4. Subjects that have had a device during the past 3 months.
    2. It is recommended that the size of the endometrium specimen be larger than 0.5 x 1 x 1 cm3. The endometrium specimen should be placed in pre-warmed (37 °C) Hank’s media containing 1% Pen/Strep and 1 μg/ml amphotrypsin B for transport. We recommend that the biopsy should be delivered to the laboratory within 2 h.
      Note: Use pre-warmed media (37 °C) for all steps.
    3. Transfer the specimen to a sterile 10 cm Petri dish using standard sterile forceps.
    4. Add fresh, pre-warmed Hank’s media containing 1% Pen/Strep and 1 μg/ml amphotrypsin B.
    5. Dissect myometrium from the endometrium and discard blood and mucus.
    6. Wash 3 times with Hank’s media (add 3-4 ml media and carefully wash tissue and discard media with a sterile pipette).
    7. Transfer the specimen to another sterile Petri dish with sterile forceps. Add isolation media (avoid letting the specimen dry out) and chop it with a sharp scalpel. Cut the specimens into 1-2 mm pieces.
      Notes:
      1. To avoid cell damage, make vertical cuts with the scalpel.
      2. Do not let the specimen dry.
    8. Transfer the cut specimen into sterile 15 ml Falcon tubes with standard sterile forceps. Add collagenase I (1 mg/ml) and incubate at 37 °C (cell incubator or water bath) for 30-45 min. Invert the falcon tube every 5 min.
    9. When cells are disassociated from the specimen (it maybe takes 2 h based on the size of the specimen (1 x 1 x 0.5 cm3), add pre-warmed (37 °C) sterile isolation media (1:2 collagenase I:isolation media) to neutralized collagenase I.
    10. Pass it through a 70 µm Falcon cell strainer once. Pass it through a 40 µm Falcon cell strainer twice to remove glandular epithelial components. Centrifuge the cells that pass through the strainer at 300 x g for 10 min. However, the centrifugation step may be performed or omitted.
    11. Slowly add the passed cells (1:1 sediment:Ficoll [100 mg/ml]) on Ficoll in a 15 ml Falcon tube at room temperature (Figure 1).


      Figure 1. hEnSC separation by Ficoll

    12. Centrifuge at 400 x g for 20 min.
    13. Discard sediment containing red blood cells and take the turbid interface phase of Ficoll and the cell culture media.
    14. Add 4 ml pre-warmed PBS to the interface phase and centrifuge at 100 x g for 10 min.
    15. Discard the supernatant, add 1 ml plating media (see Recipes) into the sediment and transfer them to T25 culture flasks. Add an additional 2.5 ml of plating media containing DMEM-F12, 15% FBS, 1% Pen/Strep. Incubate for 24 h in the cell incubator. A total volume of 3.5 ml cell culture media in T25 culture flasks for the first day can increase the frequency of cells adhering to the flask. Note that larger volumes may result in fewer cells adhering to the flask.
    16. The next day, slowly add 3 ml more of plating media and keep it in the cell incubator for 1 week. Do not shake it or disturb it each day.
    17. When cells are 90% confluent, wash them with pre-warmed PBS, discard PBS, then add 0.25% trypsin-EDTA (1 ml). Incubate in the cell incubator for 5 min. After the cells dissociate from the flask, transfer the cells to a 15 ml Falcon and add 1 ml DMEM-F12 supplanted with 10% FBS, 1% Pen/Strep. Centrifuge at 300 x g for 10 min.
    18. Add cell sediment to another T25 culture flasks (100,000 cells/ml) or T75 culture flasks (300,000 cells/ml) and for the rest of cell passages use 5 and 9 ml DMEM-F12 supplemented with 10% FBS, 1% Pene/Strep, respectively (Figure 2).
    19. After 3 passages you may scan cells by scanning electron microscopy (SEM) (Figure 3).


      Figure 2. Schematic of isolation of hEnSCs from endometrium


      Figure 3. SEM image of endometrial stem cell derived from endometrium

    Cell preparation for SEM:
    1. Cells are rinsed with PBS and then fixed in 2% glutaraldehyde for 2 h.
    2. Rinse with PBS.
    3. Incubate in 50% ethanol for 10 min.
    4. Incubate in 70% ethanol for 10 min.
    5. Incubate in 80% ethanol for 10 min.
    6. Incubate in 95% ethanol for 10 min (twice).
    7. Incubate in 100% ethanol for 10 min (twice)
    8. Freeze dry cells for 3 h.
      Note: You may sort cells as a CD146 by cell sorter (MAGS and etc…) to be sure that cell population is purified as endometrial stem cell.

  2. To isolate hEnSCs from menstrual blood, the following steps are performed (Figure 4)
    Inclusion criteria for menstrual blood (MB) are healthy females without vaginal discharge or infection, negative for HBV and HCV infection, and age ranges between 20 and 35 years old.
    1. Collect 5 ml MB with sterile Diva cups and decant into the isolation buffer 1:4 (see Recipes) in a 50 ml Falcon tube containing 2.5 µg/ml fungizone, 1% Pen/Strep and 0.5 mM EDTA in 20 ml phosphate buffered saline (PBS).
    2. Slowly add 1:1 blood sample (overlaid) to the Ficoll and centrifuge for 20 min at 400 x g.
    3. Discard sediment (pellet) containing the red blood cells and take turbid interface phase of Ficoll and cell culture media (Figure 1).
    4. Add 3 ml PBS to the interface phase and centrifuge at 100 x g for 10 min.
    5. The cell pellet is suspended in 1 ml DMEM-F12 supplemented with 10% FBS, 0.1 mM non-essential amino acids and 2 mM L-glutamine.
    6. Transfer the cell suspension to T25 culture flasks and add extra 2.5 ml of plating media. Incubate for 24 h at 37 °C in a humidified 5% CO2.
    7. Slowly add 3 ml plating media to the flask after 24 h.
      After 2 days incubation, non-adherent cells are washed away (the cell culture media is removed from the flask and 3 ml PBS is added to the flask and then 5 ml media is added to the flask). The adherent cell population (Figure 5A) and the media are replaced with media containing DMEM-F12, 1% Pen/Strep and 10% FBS.
    8. The culture media is refreshed every 3-4 days.
    9. When cells reach 90% confluence, they are passaged using trypsin/EDTA (0.25%) (Figure 5B).


      Figure 4. Isolation of hEnSCs from menstrual blood. A. Sample collection. Sample collection of menstrual blood during menstrual period in day 2 and 3 with Diva cup. This device can be inserted in vaginal canal by donors and then, blood is transported into Falcon. B. Ficoll separation.


      Figure 5. Microscopic image of MenSCs. A. 5 days after isolation; B. After passage 1.

Data analysis

To confirm that endometrial stem cells are isolated after the third passage, cells can be characterized by flow cytometry for surface markers: CD90 (mesenchymal marker), CD105 (endoglin), CD44, CD146 (endometrial stem cell markers), CD31 (Endothelial marker), CD34 (hematopoietic marker). They should be negative for these surface markers: CD31, CD34 and CD133 while are positive for these surface markers: CD90, CD105, CD44 and CD146 (Figure 1: Ebrahimi-Barough et al., 2013 and Figure 2: Mobarakeh et al., 2012).

  1. Wash cells with PBS, discard PBS, add 0.25% trypsin-EDTA, centrifuge floating cells.
  2. Wash with HBSS + 2% BSA twice and incubate with CD90, CD44, CD146, CD34, CD133 antibodies at defined concentrations recommended by their respective suppliers.
  3. Incubate for 20 min in the dark at room temperature.
  4. Asses by flow cytometry. If the stain is green, it may measure in FL1 channel and if it is a red stain, it may measure in FL2 channel. Besides, vertical column may select as a histogram. The gating strategy is based on isotype.
  5. Analyze flow cytometry data with FlowJo software.

Notes

Cells may be destroyed if cutting with the scalpel is un-carefully performed or not cut vertically. However, movement of the flask every day may decrease cell density and not allow them to completely adhere. Data analysis can be found at cited references. To isolate hEnSC from menstrual blood, you must be careful to collect the sample in a sterile manner if you do not have Diva cups. During the time you collect and transfer specimen to the laboratory, it may be kept at 4 °C in a refrigerator for 24 h. However, it is recommended that the sample should be immediately transferred to the laboratory to increase cell survival.

Recipes

  1. Isolation media for endometrium
    Pre-warmed Hank’s media supplemented with:
    5% FBS
    1% Pene/Strep
    1 μg/ml amphotrypsin B
  2. Plating media
    Pre-warmed DMEM-F12
    15% FBS
    1% Pen/Strep
    1% glutamine
    1 μg/ml amphotrypsin B
  3. Basal media for the rest of passage except first passage
    Pre-warmed DMEM-F12
    10% FBS
    1% Pene/Strep
    1% glutamine
  4. Isolation media for menstrual blood
    2.5 µg/ml fungizone
    1% Pen/Strep
    0.5 mM EDTA
    Phosphate buffered saline (PBS)
  5. Collagenase I
    1 mg collagenase is dissolved in 1 ml Hank’s media

Acknowledgments

We are very thankful to Dr. Somayeh Ebrahimi borough and Dr. Roya Karimi for their kindness help in cell isolation and characterization. Our previous works the presented protocol adapted from were funded by ‘grant numbers: 92-03-61-21215 and 92-02-61-22861’. Autours declare that there was no conflicts of interest or competing interests.

References

  1. Azedi, F., Kazemnejad, S., Zarnani, A. H., Behzadi, G., Vasei, M., Khanmohammadi, M., Khanjani, S., Edalatkhah, H. and Lakpour, N. (2014). Differentiation potential of menstrual blood- versus bone marrow-stem cells into glial-like cells. Cell Biol Int 38(5): 615-624.
  2. Azedi, F., Kazemnejad, S., Zarnani, A. H., Soleimani, M., Shojaei, A. and Arasteh, S. (2017). Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation. Mol Biol Rep 44(1): 169-182.
  3. Darzi, S., Zarnani, A. H., Jeddi-Tehrani, M., Entezami, K., Mirzadegan, E., Akhondi, M. M., Talebi, S., Khanmohammadi, M. and Kazemnejad, S. (2012). Osteogenic differentiation of stem cells derived from menstrual blood versus bone marrow in the presence of human platelet releasate. Tissue Eng Part A 18(15-16): 1720-1728.
  4. Ebrahimi-Barough, S., Kouchesfehani, H. M., Ai, J., Mahmoodinia, M., Tavakol, S. and Massumi, M. (2013). Programming of human endometrial-derived stromal cells (EnSCs) into pre-oligodendrocyte cells by overexpression of miR-219. Neurosci Lett 537: 65-70.
  5. Kazemnejad, S., Akhondi, M. M., Soleimani, M., Zarnani, A. H., Khanmohammadi, M., Darzi, S. and Alimoghadam, K. (2012). Characterization and chondrogenic differentiation of menstrual blood-derived stem cells on a nanofibrous scaffold. Int J Artif Organs 35(1): 55-66.
  6. Khanmohammadi, M., Khanjani, S., Edalatkhah, H., Zarnani, A. H., Heidari-Vala, H., Soleimani, M., Alimoghaddam, K. and Kazemnejad, S. (2014). Modified protocol for improvement of differentiation potential of menstrual blood-derived stem cells into adipogenic lineage. Cell Prolif 47(6): 615-623.
  7. Mobarakeh, Z. T., Ai, J., Yazdani, F., Sorkhabadi, S. M., Ghanbari, Z., Javidan, A. N., Mortazavi-Tabatabaei, S. A., Massumi, M. and Barough, S. E. (2012). Human endometrial stem cells as a new source for programming to neural cells. Cell Biol Int Rep (2010) 19(1): e00015.
  8. Tavakol, S., Aligholi, H., Eshaghabadi, A., Mousavi, M. M., Ai, J. and Rezayat, M. (2014a). Investigation on the motor recovery effect of a self-assembling nonofiber in the spinal cord injury model in rat. Shefaye Khatam 2: 41-46.
  9. Tavakol, S., Aligholi, H., Gorji, A., Eshaghabadi, A., Hoveizi, E., Tavakol, B., Rezayat, S. M. and Ai, J. (2014b). Thermogel nanofiber induces human endometrial-derived stromal cells to neural differentiation: In vitro and in vivo studies in rat. J Biomed Mater Res A 102(12): 4590-4597.
  10. Tavakol, S., Modarres Mousavi, S. M., Massumi, M., Amani, A., Rezayat, S. M. and Ai, J. (2015). The effect of Noggin supplementation in Matrigel nanofiber-based cell culture system for derivation of neural-like cells from human endometrial-derived stromal cells. J Biomed Mater Res A 103(1): 1-7.
  11. Tavakol, S., Mousavi, S. M. M., Tavakol, B., Hoveizi, E., Ai, J. and Sorkhabadi, S. M. R. (2017). Mechano-transduction signals derived from self-assembling peptide nanofibers containing long motif of laminin influence neurogenesis in in-vitro and in-vivo. Mol Neurobiol 54(4): 2483-2496.
  12. Tavakol, S., Musavi, S. M., Tavakol, B., Hoveizi, E., Ai, J. and Rezayat, S. M. (2016a). Noggin along with a self-assembling peptide nanofiber containing long motif of laminin induces tyrosine hydroxylase gene expression. Mol Neurobiol.
  13. Tavakol, S. Saber, R., Hoveizi, E., Aligholi, H., Ai, J. and Rezayat, S. M. (2014c). W6: Self-assembling peptide nanofiber containing biologic motif induces neural differentiation, tubulin polymerization and neurogenesis in-vitro, ex-vivo and in-vivo studies. Shefaye Khatam 2: 49-49.
  14. Tavakol, S., Saber, R., Hoveizi, E., Aligholi, H., Ai, J. and Rezayat, S. M. (2016b). Chimeric self-assembling nanofiber containing bone marrow homing peptide's motif induces motor neuron recovery in animal model of chronic spinal cord injury; an in vitro and in vivo investigation. Mol Neurobiol 53(5): 3298-3308.
  15. Tavakol, S., Saber, R., Hoveizi, E., Tavakol, B., Aligholi, H., Ai, J. and Rezayat, S. M. (2016c). Self-assembling peptide nanofiber containing long motif of laminin induces neural differentiation, tubulin polymerization, and neurogenesis: in vitro, ex vivo, and in vivo studies. Mol Neurobiol 53(8): 5288-5299.

简介

人类子宫内膜干细胞/基质细胞(hEnSCs)是从子宫内膜或经血中分离出来的,在组织工程和细胞治疗中被公认为有价值的细胞类型。 此外,在细胞表面具有CD90(间充质标记),CD105(内皮糖蛋白),CD44,CD146(子宫内膜干细胞标记)和缺乏CD31(内皮标记),CD34(造血标记)和CD133的hEnSCs是新的 间充质干细胞/基质细胞的来源。 此外,这些细胞可以封装成自组装肽纳米纤维作为三维支架应用于治疗神经退行性疾病。 在这里,我们描述了从子宫内膜或经血中分离hEnSC的方案。

【背景】正在研究细胞替代治疗,作为治疗神经退行性疾病(如阿尔茨海默病,中风和脊髓损伤)的新策略(Tavakol等人,2014b; 2015; 2016a和2016c)。子宫内膜干细胞/基质细胞(hEnSCs)可以从经血(Azedi等人,2014和2017)和子宫内膜分离。这些细胞具有脂肪形成(Khanmohammadi等人,2014),成骨(Darzi等人,2012)和软骨形成(Kazemnejad等人, >,2012)分化潜力。此外,这些细胞可以与支架组合用作组织工程应用中智能细胞支架的成分(Tavakol等人,2014a; 2016b和2017)。值得注意的是,诸如外来体,微泡和其他组分的细胞区室被认为比使用全细胞的治疗更安全。 hEnSC是细胞治疗的有吸引力的候选者,因为它们是免疫抑制性的,并且具有很高的克隆性(1.25%)潜力。它们也比其他细胞类型有优势。 hESC比胚胎干细胞更适合于细胞治疗,因为hESC不发生畸胎瘤。与间充质干细胞不同,hEnSC不会降低老年人的增殖能力(Ebrahimi-Barough等人,2013)。此外,hEnSC的另一个有利特征是它们可以被封装成自组装肽纳米纤维并分化成神经元细胞(Tavakol等,2014c; 2016a; 2016c和2017)。该协议描述了一个简单的策略,可以从经血和子宫内膜获得更高产量的hEnSC,用于体内和体内,可用于许多研究。

关键字:子宫内膜干细胞, 子宫内膜间质细胞, 经血, 细胞分离, 神经分化, 自组装肽纳米纤维, 3D细胞培养

材料和试剂

  1. 移液枪头:结晶,黄色和蓝色(Gilson,产品编号:JHA004,JHA005和JHA007)
  2. SPL细胞培养皿,90×15mm(SPL Life Sciences,目录号:11090)
  3. 猎鹰管15毫升(SPL生命科学,目录号:50115)
  4. PluriStrainer70μm(Pluriselect,目录号:43-50070)
  5. Falcon细胞过滤器40微米(Corning,Falcon ,目录号:352340)
  6. T25培养瓶(SPL Life Sciences,目录号:70125)
  7. T75培养瓶(SPL Life Sciences,目录号:70175)
  8. Diva杯(Diva国际,Lunette,芬兰)
  9. 猎鹰管50毫升(SPL生命科学,目录号:50050)
  10. 子宫内膜
  11. 月经血液
  12. Ficoll-Hypaque(GE Healthcare,Amersham,目录号:17-5442-02)
  13. FITC-结合的抗CD105(Abcam,目录号:ab18278)
  14. 别藻蓝素结合的抗CD44抗体(Abcam,目录号:ab81424)
  15. 抗CD34抗体[EP373Y](Abcam,目录号:ab81289)
  16. 抗CD133抗体(Abcam,目录号:ab19898)
  17. 青霉素/链霉素(Pen / Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  18. 胰蛋白酶B溶液(Sigma-Aldrich,目录号:A2942)
  19. 胶原酶I(Thermo Fisher Scientific,Gibco TM,目录号:17100017)
  20. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:806552)
  21. DMEM-F12(Thermo Fisher Scientific,Gibco TM,产品目录号:11330057)
  22. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  23. 胰蛋白酶-EDTA 0.25%(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  24. 戊二醛,25%水溶液(Merck,目录号:354400)
  25. 乙醇(Merck,目录号:818760)
  26. Fungizone(Thermo Fisher Scientific,Gibco TM,目录号:15290018)
  27. 乙二胺四乙酸四钠盐脱水物(EDTA)(Sigma-Aldrich,目录号:E6511)
  28. 非必需氨基酸(Thermo Fisher Scientific,Gibco TM,目录号:11140035)
  29. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  30. Hank's平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:24020117)
  31. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2058)
  32. 用于子宫内膜的隔离介质(见食谱)
  33. 电镀媒体(见食谱)
  34. 基本媒体的其余部分,除了第一段(请参阅食谱)
  35. 隔离媒体经血(见食谱)
  36. 胶原酶我(见食谱)

设备

  1. 手术刀(精细科学工具,目录号:10011-00,10003-12)
  2. 标准无菌镊子(BYDAND,目录号:BSU101)
  3. 无菌移液器(SPL LIFE SICENCES,产品目录号:91005)
  4. 37℃,5%CO 2细胞培养箱(New Brunswic Scientific,型号:CO-150)。
  5. 离心机(Eppendorf,型号:5810 R)
  6. 倒置显微镜(奥林巴斯,型号:IX51)
  7. 冰箱(Pars,型号:1300)

软件

  1. FlowJo 7.6.1软件(FlowJo,LLC; https://www.flowjo.com

程序

注意:子宫内膜活检必须由专家外科医生按照当地的道德操守政策和指引进行。

  1. 为了从子宫内膜分离hEnSC,执行以下步骤
    1. 子宫内膜标本活检
      活检应取自:
      1. 健康的女性
      2. 年龄:20-35岁之间
      3. 时间:子宫内膜活检和经血分别在月经周期19-24和2-4分离。
      活组织检查不应该从以下方面获得:
      1. 有非子宫内膜良性病理状况,如息肉,增生或癌症的受试者。
      2. 在活组织检查之前的几个月内服用了外源激素如GnRH,黄体酮和其他激素的受试者。

      3. 子宫内膜异位症或任何病理状况的子宫
      4. 在过去3个月内曾使用过设备的受试者。
    2. 建议子宫内膜样本大小大于0.5×1×1 cm3。将子宫内膜标本置于预热的(37℃)汉克氏培养基(含有1%Pen / Strep和1μg/ ml的两性胰蛋白酶B)中运输。我们建议活检应在2小时内送到实验室。
      注意:所有步骤均使用预热介质(37°C)。

    3. 使用标准的无菌镊子将样本转移到无菌的10厘米培养皿中
    4. 加入含有1%Pen / Strep和1μg/ ml amphotrypsin B的新鲜预热Hank's培养基。

    5. 从子宫内膜中解剖出子宫肌层,丢弃血液和粘液
    6. 用Hank's培养基清洗3次(加入3-4ml培养基,小心地清洗组织并用无菌移液管丢弃培养基)。
    7. 将样本转移到另一个无菌培养皿,用无菌镊子。加入隔离介质(避免让样本变干),并用锋利的手术刀切碎。切成1-2毫米的标本。
      注意:
      1. 为避免细胞损伤,请用手术刀垂直切割。
      2. 不要让标本干燥。
    8. 将切下的标本转移到带有标准无菌镊子的无菌15ml Falcon管中。加入胶原酶I(1mg / ml)并在37℃(细胞培养箱或水浴)中孵育30-45分钟。
      每5分钟翻转猎鹰管
    9. 当细胞与样品分离时(根据样品的大小(1 x 1 x 0.5 cm 3)可能需要2 h),加入预热的(37°C)无菌隔离介质( 1:2胶原酶I:分离介质)中和胶原酶I.
    10. 将其通过70μmFalcon细胞过滤器一次。将其通过40μmFalcon细胞过滤器两次以去除腺上皮组分。离心通过过滤器的细胞在300克xg 10分钟。然而,可以执行或省略离心步骤。
    11. 在室温下在15ml Falcon管中的Ficoll缓慢加入通过的细胞(1:1沉淀物:Ficoll [100mg / ml])(图1)。


      图1. Ficoll的hEnSC分离

    12. 在400×g离心20分钟。
    13. 丢弃含有红细胞的沉淀物,并进入Ficoll的浑浊界面阶段和细胞培养基。
    14. 添加4毫升预热的PBS到界面阶段并在100×g下离心10分钟。
    15. 弃上清,加入1毫升电镀媒体(见食谱)沉淀物,并转移到T25培养瓶。加入另外2.5ml含有DMEM-F12,15%FBS,1%Pen / Strep的平板培养基。在细胞培养箱中孵育24小时。 T25培养瓶中第一天总体积为3.5ml的细胞培养基可以增加细胞粘附于培养瓶的频率。请注意,较大的体积可能会导致更少的细胞粘附在烧瓶上。
    16. 第二天,慢慢加入3毫升以上的平板培养基,并保存在细胞培养箱中1周。不要每天摇晃或打扰。
    17. 当细胞达到90%融合时,用预热的PBS洗涤,丢弃PBS,然后加入0.25%胰蛋白酶-EDTA(1ml)。在细胞培养箱中孵育5分钟。细胞从培养瓶中解离后,将细胞转移到15ml Falcon中,加入1ml代替10%FBS,1%Pen / Strep的DMEM-F12。
      在300×g离心10分钟
    18. 将细胞沉淀物加入到另外的T25培养瓶(100,000个细胞/ ml)或T75培养瓶(300,000个细胞/ ml)中并且对于其余的细胞传代使用补充有10%FBS,1%Pene / Strep的5和9ml DMEM-F12 (图2)。
    19. 3次传代之后,您可以通过扫描电子显微镜(SEM)(图3)扫描细胞。


      图2.从子宫内膜分离hEnSC的示意图


      图3.来自子宫内膜的子宫内膜干细胞的SEM图像

    SEM的细胞准备
    1. 细胞用PBS冲洗,然后在2%戊二醛中固定2小时。
    2. 用PBS冲洗。

    3. 在50%乙醇中孵育10分钟
    4. 在70%的乙醇中孵育10分钟。
    5. 在80%乙醇中孵育10分钟。
    6. 在95%乙醇中孵育10分钟(两次)。
    7. 在100%乙醇中孵育10分钟(两次)
    8. 将干细胞冷冻3小时。
      注:您可以通过细胞分选仪(MAGS等)将细胞分选为CD146,以确保细胞群被纯化为子宫内膜干细胞。

  2. 为了从经血中分离hEnSCs,执行以下步骤(图4) 女性月经血(MB)的入选标准是没有阴道分泌物或感染的健康女性,HBV和HCV感染阴性,年龄在20-35岁之间。
    1. 用无菌Diva杯收集5毫升的MB,并在20毫升磷酸盐缓冲盐水(50毫升含有2.5微克/毫升fungizone,1%笔/链霉素和0.5毫米EDTA的50毫升猎鹰管1:4(见食谱) PBS)。
    2. 缓慢加入1:1血液样品(覆盖)到Ficoll中,并在400×g离心20分钟。
    3. 丢弃含有红细胞的沉淀物(颗粒)并取Ficoll和细胞培养基的浑浊界面相(图1)。
    4. 将3ml PBS加入界面相,并在100gxg离心10分钟。
    5. 将细胞沉淀物悬浮于1ml补充有10%FBS,0.1mM非必需氨基酸和2mM L-谷氨酰胺的DMEM-F12中。
    6. 将细胞悬液转移到T25培养瓶中并加入额外的2.5ml电镀培养基。在37℃潮湿的5%CO 2中孵育24小时。

    7. 24小时后,慢慢加3 ml电镀液 孵育2天后,洗去未贴壁细胞(将细胞培养基从烧瓶中取出并将3ml PBS加入烧瓶中,然后将5ml培养基加入烧瓶中)。用含有DMEM-F12,1%Pen / Strep和10%FBS的培养基代替贴壁细胞群体(图5A)和培养基。
    8. 培养基每3-4天更新一次。
    9. 当细胞达到90%汇合时,它们使用胰蛋白酶/ EDTA(0.25%)传代(图5B)。


      图4.从经血中分离hEnSCs A.样品收集。第二天和第三天月经期间的月经血样本采用歌德杯。这个装置可以被捐献者插入阴道,然后血液被运送到猎鹰。 B. Ficoll分离。


      图5. MenSCs的显微图像。 :一种。隔离后5天; B.通过1.

数据分析

为了证实子宫内膜干细胞在第三次传代后被分离,可以通过流式细胞术表征细胞表面标志物:CD90(间充质标志物),CD105(内皮糖蛋白),CD44,CD146(子宫内膜干细胞标志物),CD31(内皮标志物) ,CD34(造血标记)。它们对于这些表面标记物应该是阴性的:CD31,CD34和CD133,而对于这些表面标记物是阳性的:CD90,CD105,CD44和CD146(图1:Ebrahimi-Barough等人, 2:Mobarakeh et al。,2012)。

  1. 用PBS洗涤细胞,丢弃PBS,加入0.25%胰蛋白酶-EDTA,离心浮游细胞。
  2. 用HBSS + 2%BSA洗涤两次,并以各自供应商推荐的确定浓度与CD90,CD44,CD146,CD34,CD133抗体一起温育。

  3. 在室温下黑暗中孵育20分钟
  4. 通过流式细胞仪进行分析。如果污迹为绿色,则可以在FL1通道中测量,如果是红色污迹,则可以在FL2通道中测量。此外,垂直列可以选择为直方图。门控策略是基于同种。
  5. 使用FlowJo软件分析流式细胞术数据。

笔记

如果用手术刀切割不慎或不垂直切割,细胞可能会被破坏。但是,每天移动烧瓶可能会降低细胞密度并且不能使它们完全粘附。数据分析可以在引用的参考文献中找到。为了从经血中分离hEnSC,如果您没有Diva杯,您必须小心地以无菌方式收集样品。在您收集标本并将其转移到实验室的过程中,可能需要在4°C的冰箱中保存24小时。但是,建议将样品立即转移到实验室以增加细胞存活。

食谱

  1. 子宫内膜隔离介质
    预热汉克的媒体补充:
    5%FBS
    1%Pene / Strep
    1μg/ ml的两性蛋白B
  2. 电镀媒体
    预热的DMEM-F12
    15%FBS
    1%Pen / Strep
    1%谷氨酰胺
    1μg/ ml的两性蛋白B
  3. 基本媒体的其余部分,除了第一次通过
    预热的DMEM-F12
    10%FBS
    1%Pene / Strep
    1%谷氨酰胺
  4. 经血液隔离介质
    2.5微克/毫升fungizone
    1%Pen / Strep
    0.5 mM EDTA
    磷酸盐缓冲盐水(PBS)
  5. 胶原酶I
    1毫克胶原酶溶解在1毫升的汉克氏媒体

致谢

我们非常感谢Somayeh Ebrahimi博士和Roya Karimi博士对细胞分离和鉴定的帮助。我们以前的作品改编自提出的协议由“资助号码:92-03-61-21215和92-02-61-22861”资助。 Autours宣称没有利益冲突或利益冲突。

参考

  1. Azedi,F.,Kazemnejad,S.,Zarnani,A. H.,Behzadi,G.,Vasei,M.,Khanmohammadi,M.,Khanjani,S.,Edalatkhah,H.和Lakpour,N.(2014年)。 经血与骨髓干细胞向神经胶质样细胞的分化潜能 Cell Biol Int 38(5):615-624。
  2. Azedi,F.,Kazemnejad,S.,Zarnani,A. H.,Soleimani,M.,Shojaei,A。和Arasteh,S.(2017)。 经血与骨髓干细胞在神经分化中的比较能力 Mol Biol Rep 44(1):169-182。
  3. Darzi,S.,Zarnani,A. H.,Jeddi-Tehrani,M.,Entezami,K.,Mirzadegan,E.,Akhondi,M.M。,Talebi,S.,Khanmohammadi,M.和Kazemnejad,S.(2012)。 在存在人血小板释放物的情况下经血与骨髓衍生的干细胞的成骨分化< / a> Tissue Eng Part A 18(15-16):1720-1728。
  4. Ebrahimi-Barough,S.,Kouchesfehani,H.M.,Ai,J.,Mahmoodinia,M.,Tavakol,S。和Massumi,M。(2013)。 人类子宫内膜来源的间质细胞(EnSCs)通过过表达miR- 219. Neurosci Lett 537:65-70。
  5. Kazemnejad,S.,Akhondi,M. M.,Soleimani,M.,Zarnani,A. H.,Khanmohammadi,M.,Darzi,S.和Alimoghadam,K.(2012)。 纳米纤维支架上经血源性干细胞的表征和成软骨分化 < Int J Artif Org ans 35(1):55-66。
  6. Khanmohammadi,M.,Khanjani,S.,Edalatkhah,H.,Zarnani,A. H.,Heidari-Vala,H.,Soleimani,M.,Alimoghaddam,K.和Kazemnejad,S.(2014)。 经改进的方案,改善经血源性干细胞分化成脂肪细胞的能力 Cell Prolif 47(6):615-623。
  7. Mobarakeh,Z.T.,Ai,J.,Yazdani,F.,Sorkhabadi,S.M.,Ghanbari,Z.,Javidan,A.N。,Mortazavi-Tabatabaei,S.A.,Massumi,M.and Barough,S.E。(2012) 人类子宫内膜干细胞作为编程神经细胞的新来源 Cell Biol Int Rep(2010)19(1):e00015。
  8. Tavakol,S.,Aligholi,H.,Eshaghabadi,A.,Mousavi,M. M.,Ai,J.和Rezayat,M.(2014a)。 大鼠脊髓损伤模型中自组装非纤维的运动恢复效应的研究。 Shefaye Khatam 2:41-46。
  9. Tavakol,S.,Aligholi,H.,Gorji,A.,Eshaghabadi,A.,Hoveizi,E.,Tavakol,B.,Rezayat,S.M。和Ai,J.(2014b)。 热凝胶纳米纤维诱导人子宫内膜来源的基质细胞进行神经分化:体外和 in vivo 在大鼠中的研究。 J Biomed Mater Res A 102(12):4590-4597。
  10. Tavakol,S.,Modarres Mousavi,S. M.,Massumi,M.,Amani,A.,Rezayat,S.M。和Ai,J。(2015)。 Noggin在基于Matrigel纳米纤维的细胞培养系统中补充神经样细胞人类子宫内膜来源的基质细胞。 J Biomed Mater Res A 103(1):1-7。
  11. Tavakol,S.,Mousavi,S. M. M.,Tavakol,B.,Hoveizi,E.,Ai,J.和Sorkhabadi,S. M. R.(2017)。 源自含有长层粘连蛋白基序的自组装肽纳米纤维的机械转导信号影响神经发生>体外和体内。 Mol Neurobiol 54(4):2483-2496。
  12. Tavakol,S.,Musavi,S.M.,Tavakol,B.,Hoveizi,E.,Ai,J.and Rezayat,S.M。(2016a)。 Noggin和含有长链基因层粘连蛋白的自组装肽纳米纤维诱导酪氨酸羟化酶基因表达。 / Mol> Neurobiol 。
  13. Tavakol,S. Saber,R.,Hoveizi,E.,Aligholi,H.,Ai,J.和Rezayat,S.M。(2014c)。 W6:自组装肽纳米纤维含有生物motif在体外诱导神经分化,微管蛋白聚合和神经发生体外研究 Shefaye Khatam 2:49-49。
  14. Tavakol,S.,Saber,R.,Hoveizi,E.,Aligholi,H.,Ai,J.和Rezayat,S.M。(2016b)。含有骨髓归巢肽基序的嵌合自组装纳米纤维在慢性脊髓动物模型中诱导运动神经元恢复脊髓损伤; 和 in vivo 调查。Mol Neurobiol 53(5):3298-3308。
  15. Tavakol,S.,Sabre,R.,Hoveizi,E.,Tavakol,B.,Aligholi,H.,Ai,J.和Rezayat,S.M。(2016c)。 含有层粘连蛋白长基序的自组装肽纳米纤维诱导神经分化,微管蛋白聚合和神经发生:体外研究 和体内研究。 Mol Neurobiol 53(8):5288 -5299。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tavakol, S., Azedi, F., Hoveizi, E., Ai, J. and Joghataei, M. (2018). Human Endometrial Stem Cell Isolation from Endometrium and Menstrual Blood. Bio-protocol 8(2): e2693. DOI: 10.21769/BioProtoc.2693.
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