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Jan 2020

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Generation of the Compression-induced Dedifferentiated Adipocytes (CiDAs) Using Hypertonic Medium
使用高渗培养基压缩诱导的去分化脂肪细胞(CiDAs)的产生   

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Abstract

Current methods to obtain mesenchymal stem cells (MSCs) involve sampling, culturing, and expanding of primary MSCs from adipose, bone marrow, and umbilical cord tissues. However, the drawbacks are the limited numbers of total cells in MSC pools, and their decaying stemness during in vitro expansion. As an alternative resource, recent ceiling culture methods allow the generation of dedifferentiated fat cells (DFATs) from mature adipocytes. Nevertheless, this process of spontaneous dedifferentiation of mature adipocytes is laborious and time-consuming. This paper describes a modified protocol for in vitro dedifferentiation of adipocytes by employing an additional physical stimulation, which takes advantage of augmenting the stemness-related Wnt/β-catenin signaling. Specifically, this protocol utilizes a polyethylene glycol (PEG)-containing hypertonic medium to introduce extracellular physical stimulation to obtain higher efficiency and introduce a simpler procedure for adipocyte dedifferentiation.

Keywords: Mesenchymal stem cells (间充质干细胞), Dedifferentiation (去分化), Adipocytes (脂肪细胞), Compression (压缩), Wnt/β-catenin signaling (Wnt /β连环蛋白信号)

Background

Adipose tissue currently is one of the most appealing sources of mesenchymal stem cells (MSCs), due to its large abundance and relatively less-invasive harvest methods (Shen et al., 2011; González-Cruz et al., 2012; Konno et al., 2013). Adipose-derived MSCs, that isolated from the stromal-vascular fraction of subcutaneous adipose tissue, have been demonstrated to display multilineage potentials both in vitro and in vivo (Anghileri et al., 2008; González et al., 2009; Gonzalez-Rey et al., 2010; Jumabay et al., 2010; Mao et al., 2017 and 2019; Darnell et al., 2018). To isolate adipose-derived MSCs, the widely-used method is to dissect the stromal-vascular fraction from the adipose tissue, and then sort the MSCs by either fluorescence-activated cell sorting (FACS) or culture selection (Aronowitz et al., 2015; Raposio et al., 2017; Gentile et al., 2019). However, heterogeneous groups of cells are contained in a stromal-vascular fraction of adipose tissue, and limited cell markers are available for MSCs selection; these make it difficult to purify adipose-derived MSCs (Gimble et al., 2011; González-Cruz et al., 2012; Konno et al., 2013).


Alternatively, the adipocytes, rather than the other types of cells in adipose tissue, can spontaneously dedifferentiate into multipotent mesenchymal cells named the dedifferentiated fat (DFAT) cells during in vitro culturing (Sugihara et al., 1986; Shen et al., 2011; Taniguchi et al., 2016). Because of the multipotency of the DFAT cells and the large abundance of the mature adipocytes, the DFAT cells have been regarded as an ideal source for human postnatal mesenchymal multipotent stem cells (Matsumoto et al., 2008; Shen et al., 2011; Côté et al., 2019). However, the current ceiling culturing for adipocyte dedifferentiation requires a long duration (typically 4 weeks) to enable the adipocytes to spontaneously lose all obvious lipid droplets (Lessard et al., 2015; Taniguchi et al., 2016). Thus, further increasing the efficiency of adipocyte dedifferentiation and shortening its processing time is attractive for its wider applications.


Adipocytes and adipose progenitor cells are also important components in tumor microenvironments (Chandler et al., 2012; Seo et al., 2015; Ling et al., 2020). Recent studies revealed that the dedifferentiation of adipocytes occurred during tumor development, which might be attributed to the activated Wnt signaling (Gustafson and Smith, 2010; Bochet et al., 2013) and Notch signaling (Bi et al., 2016). Recent studies also revealed that the dedifferentiation of adipocytes could occur in vivo in mice models (Bochet et al., 2013; Liao et al., 2015; Wang et al., 2018). Tumor progression also largely alters the local physical microenvironments, including elevated osmotic pressure, increased compressive force, and matrix stiffening (Nia et al., 2020). These physical cues largely influence the cell fates of both adipose stromal cells and cancer cells (Guo et al., 2017; Li et al., 2019 and 2020a; Han et al., 2020). Indeed, our recent study reported that the generation of osmotic stress in vitro to mimic the elevated osmolarity in in vivo tumors could also induce the dedifferentiation of adipocytes (Li et al., 2020b). Consistently, another study also reported that a tough implant in vivo drove the dedifferentiation of the local surrounding adipocytes (Ma et al., 2019). Thus, these studies inspired us to develop an alternative protocol to generate multipotent mesenchymal cells by mechanically dedifferentiating adipocytes.


The protocol described here includes the experimental set-ups to induce and verify the reprogramming of adipocytes into multipotent mesenchymal cells using our hypertonic dedifferentiation medium. We also include the procedures to generate adipocytes from preadipocytes or mesenchymal stem cells, and the differentiation assays to confirm the multilineage potentials of the CiDAs.


Materials and Reagents

  1. Reagents

    1. Minimum Essential Medium Eagle Alpha Modification media (Sigma-Aldrich, catalog number: M8042)

    2. Fetal bovine serum (Gibco, catalog number: 10-082-147)

    3. Penicillin/streptomycin (Gibco, catalog number: 15140148)

    4. Polyethylene glycol 300 (Sigma-Aldrich, catalog number: 8.07484)

    5. KnockOut Serum Replacement (Gibco, catalog number: 10828-028)

    6. Preadipocyte Growth Medium-2 (Lonza, catalog number: PT-8202)

    7. SingleQuots (Lonza, catalog number: PT-9502)

    8. Paraformaldehyde (VWR, catalog number: IC0219998380)

    9. PBS (Sigma-Aldrich, catalog number: P5119)

    10. Triton-X-100 (Sigma-Aldric, catalog number: X100)

    11. Oil Red O (Sigma-Aldrich, catalog number: O0625)

    12. DMEM (Sigma-Aldrich, Brand, catalog number: D5546)

    13. Horse serum (Gibco, catalog number: 26050070)

    14. Dexamethasone (Sigma-Aldrich, catalog number: D4902)

    15. Hydrocortisone (Sigma-Aldrich, catalog number: H0888)

    16. Hydrogen peroxide (Sigma-Aldrich, catalog number: H1009)

    17. Anti-MyoD1 (Abcam, catalog number: ab16148)

    18. Donkey anti-Rabbit Alexa 488 (Invitrogen, catalog number: R37118)

    19. β-glycerophosphate (Sigma-Aldrich, catalog number: G9422)

    20. L-ascorbic acid (Sigma-Aldrich, Brand, catalog number: A4403)

    21. ELF-97 (Invitrogen, catalog number: E6588)

    22. TGF-β (R&D Systems, catalog number: 240-B)

    23. Anti-aSMA (Abcam, catalog number: ab5694)

    24. DAPI (Thermo Scientific, Brand, catalog number: 62248)

    25. Trypsin (2.5%) (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046)


  2. Cell culture plasticware

    1. T75 and/or T25 flasks (Corning, catalog numbers: 430641U for T75 and 3056 for T25)

    2. Centrifuge tubes (15 ml; 50 ml, Corning, catalog numbers: 430790; 430828)

    3. Cryovials (STARLAB, catalog number: E3110-6122)

    4. Pipette tips (TipOne, STARLAB, catalog numbers: S1111-3700; S1111-1706; S1111-6701)

    5. 35-mm cell culture dish (Thermo Fisher Scientific, catalog number: 153066)

    6. 6-well plates (Corning, Falcon®, catalog number: 353934)

    7. 100 mm cell culture dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150464)

Equipment

  1. Centrifuge (Eppendorf, model: 5810)

  2. Bright-LineTM Hemacytometer (Sigma-Aldric, catalog number: Z359629)

  3. Water bath (Thermo Scientific, catalog number: TSCIR19)

  4. Humidified incubator at 37 °C, 5% CO2 (Thermo Fisher Scientific, Heraeus, model: HeracellTM 150)

  5. Leica TCS SP8 Confocal Microscope (Leica)

  6. ZEISS Axio Zoom V16 microscope (ZEISS)

  7. Xenon Arc Lamp (ZEISS)

  8. Hamamatsu Orca Flash 4.0 V3 (Scientifica)

  9. Aspirator (Dry vacuum pump/compressor, Welch Vacuum - Gardner Denver, model: 2511)

Software

  1. ImageJ (https://imagej.nih.gov/ij/)

  2. LAS X (Leica Microsystems, Mannheim, Germany)

  3. HCImage (http://www.hamamatsu.com/)

Procedure

  1. Cell culture (Figure 1, step 1)

    1. Purchase clonally derived mouse MSCs (OP9) from the American Type Cell Culture (ATCC).

    2. Expand MSCs (OP9) subconfluently in Minimum Essential Medium Eagle Alpha Modification media supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin (complete MEM) in the condition of 5% CO2, 37 °C, and 95% humidity.

    3. Assess cell viability using calcein acetoxymethyl and ethidium homodimer-1 (Invitrogen, Eugene, OR) or trypan blue exclusion (Beckman Coulter).

    4. Purchase subcutaneous primary human preadipocytes from Lonza.

    5. Culture primary human preadipocytes at subconfluence in Preadipocyte Growth Medium-2 (Lonza) in the condition of 5% CO2, 37 °C, and 95% humidity, following the manufacturer’s instructions.


  2. Generation of adipocytes from preadipocytes or mesenchymal stem cells (Figure 1, step 2)

    1. Induce adipogenesis of OP9 mMSCs by supplementing cells with MEM (Gibco) containing 15% KnockOut Serum Replacement (Gibco).

    2. Induce adipogenesis of human preadipocytes by culturing cells in Preadipocyte Growth Medium-2 (Lonza) supplemented with SingleQuots (Lonza) consisting of insulin, dexamethasone, indomethacin, and isobutyl-methylxanthine, as per the manufacturer’s instructions.


  3. Sort adipocytes from the mixed cell population

    1. Trypsinize the mixed cell population after adipogenesis induction.

    2. Transfer the cell suspension into a 15 ml centrifuge tube, and centrifuge at a low speed (150 × g, 5 min). The differentiated adipocytes are then floating on the top layer of the medium in the centrifuge tube due to their lower density as compared to the culture medium (Figure 1, step 3).

    3. Take up only the differentiated adipocytes from the top layer in the centrifuge tube, and seed 105 cells per culture flask (Falcon 3012; 25 cm2). Incubate cells at 37 °C in a medium consisting of Minimum Essential Medium Eagle Alpha Modification media supplemented with 20% fetal bovine serum.

    4. Completely fulfill the flask with medium to provide the mixed cells with an air-free environment (Figure 1, step 4).

    5. Turn the flask upside down on the first day during culturing, allowing the adipocytes to float up in the medium and adhere to the top inner surface (ceiling surface) of the flasks (Figure 1, step 5).

    6. Turn the flask back after cells are fully attached (in most cases 1 day is enough, not exceeding 2 days) so that cells are back on the bottom of the flask again (Figure 1, step 6).

    7. To obtain purified and monodispersed adipocyte population, sufficiently digest and pipet the cells.

    8. Gently wash away the medium and the residual unattached cells.

    9. Culture adipocytes with 5 ml medium contained in one Flask in the condition of 5% CO2, 37 °C, and 95% humidity. There are also some numbers of undifferentiated mesenchymal stem cells or preadipocytes attached to the ceiling surface of the flask. Without the supplement of a medium, these cells are then exposed directly to the air and die shortly (Figure 1, step 7).



      Figure 1. Schematic illustration of the procedure to generate compression-induced dedifferentiation of adipocytes. 1. Mesenchymal stem cells or adipocyte progenitors are homogeneously seeded in tissue culture plate (Step A). 2. Induction of adipogenesis of MSCs or adipocyte progenitors using adipogenesis medium (Step B). 3. Trypsinize the cells and sort generated adipocytes by density (Steps C1-C2). 4. Fulfill the cell culture flask using cell medium and the floating adipocytes (Steps C3-C4). 5. Turn the cell culture flask upside to allow adipocytes attaching to the bottom of the flask by ceiling culturing (Step C5). 6. Turn over the cell culture flask after the adipocytes fully attached to the bottom of flask (Step C6). 7. Induction of dedifferentiation of adipocyte using osmotic compression (Step D).


  4. Induction of compression-induced dedifferentiation of adipocytes

    1. Aspire and remove half of the culture medium (2.5 ml) from the flask.

    2. Add 2.5 ml hypertonic dedifferentiation medium into the flask [Minimum Essential Medium Eagle Alpha Modification media supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin, and 4% PEG-300 (MW: 300, v/v ratio)]. Avoid shaking the flask, and allow the hypertonic medium to slowly diffuse and mix with a residual culturing medium in the flask. Culture the cells in the condition of 5% CO2, 37 °C, and 95% humidity.

    3. Exchange the hypertonic dedifferentiation medium every 3 days. Aspire and remove 4 ml of the medium from the flask, and refill with 4 ml hypertonic dedifferentiation medium [Minimum Essential Medium Eagle Alpha Modification media supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin, and 2% PEG-300 (MW: 300, v/v ratio)].

    4. Avoid complete removal of the medium and avoid any shear that could be applied to adipocytes. Because of the fragile property of the adipocytes and their contained lipid droplets, any shear or rapid osmotic stress changes may damage the cells.

    5. Image the cultured adipocytes during culturing. In 10 days, we likely observe half of the population of the adipocytes transiting and dedifferentiating to mesenchymal stromal cell-like cells (Figure 2). Other adipocytes remain large lipid droplets, many of which would not undergo dedifferentiation.



      Figure 2. Dedifferentiation of adipocytes before and after 9 days of compression treatment. A. Adipocytes with cellular lipid droplets before osmotic compression-induced dedifferentiation. B. Anticipated results of CiDAs. The red arrows indicate the regions CiDAs are located, while some other adipocytes are remaining their lipid droplets. Scale bar, 100 μm. (Step D)


    6. Trypsinize all the cells and transfer them to a 15 ml centrifuge tube. Centrifuge the cells at a speed of 150 × g for 5 min.

    7. Remove the top layer residual adipocytes and the supernatant. Resuspend the CiDAs in MSCs expansion medium (Minimum Essential Medium Eagle Alpha Modification media supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin) and seed the cells back to a flask with a density of 105 cells per culture flask. Culture the cells in the condition of 5% CO2, 37 °C, and 95% humidity.

    8. Exchange the expansion medium every other day, and keep doing this up to 2 weeks until the CiDAs reaching 80% confluence of the flask surface.


  5. Inducing osteogenesis of CiDAs to test the multilineage potential of CiDAs

    1. Seed the harvested CiDAs in the wells of 6-well plates with a density of 3 × 105 cells per well, and culture them with MSCs expansion medium (5% CO2, 37 °C, and 95% humidity) until CiDAs reaching more than 90% confluence in the well.

    2. Exchange the expansion medium with osteogenic medium (complete DMEM supplemented with 10 mM β-glycerophosphate and 250 µM L-ascorbic acid). Cycle the osteogenic medium every 2 days.

    3. To test ALP activity, fix CiDAs after 6 days of culturing in an osteogenic medium. Permeabilize CiDAs with Triton X-100. Stain the fixed CiDAs with ELF-97 (Thermo Fisher Scientific), following the manufacturer’s instructions.

    4. Image the stained CiDAs using epifluorescence microscopy, consisting of a Xenon lamp, an Axio Zoom V16 microscope, and Hamamatsu Flash 4.0 v3. Anticipate observing over 50% of the cells are positive with the blue color of ALP staining (Figure 3A). The positive ratio of calcium deposition can be accessed using ImageJ, which is defined by the number of pixels positive with red color divided by the total numbers of pixels of the image.

    5. To test mineral deposition of osteogenesis of CiDAs, firstly aspirate the medium from the well. Then, fix the cells in ice-cold 70% ethanol for 5 min at room temperature. Aspirate alcohol and rinse cells twice with DI water (5 min each time). Aspirate the water and add 1 ml 2% Alizarin Red S solution, which is adjusted to a pH value of 4.1-4.3 with ammonium hydroxide. Incubate the well plate at room temperature for 3 min. Aspirate Alizarin Red S solution and wash the wells five times with 2 ml water.

    6. Image the stained CiDAs using epifluorescence microscopy, consisting of a Xenon lamp, an Axio Zoom V16 microscope, and Hamamatsu Flash 4.0 v3. Anticipate to observe over 50% of the surface is positive with the red color of calcium deposition staining (Figure 3B).


  6. Inducing adipogenesis of CiDAs to test the multilineage potential of CiDAs

    1. Seed the harvested CiDAs in the wells of 6-well plates with a density of 3 x 105 cells per well, and culture them with MSCs expansion medium (5% CO2, 37 °C, and 95% humidity) until CiDAs reaching more than 90% confluence in the well.

    2. If the CiDAs is originating from mouse MSCs (OP9), exchange the expansion medium with an adipogenic medium (MEM (Gibco) containing 15% KnockOut Serum Replacement (Gibco)). Cycle the osteogenic medium every 2 days.

    3. If the CiDAs is originating from human primary preadipocytes, exchange the expansion medium with an adipogenic medium (Preadipocyte Growth Medium-2 (Lonza) supplemented with SingleQuots (Lonza) consisting of insulin, dexamethasone, indomethacin, and isobutyl-methylxanthine). Cycle the osteogenic medium every 2 days.

    4. To test lipid droplet accumulation after 10 days of incubation, fix CiDAs with 4% paraformaldehyde (Thermo Fisher Scientific) in phosphate-buffered saline (PBS) (Gibco) with 0.1% Triton X-100 (Sigma-Aldrich) for 30 min at 25 °C. Wash CiDAs with PBS 3 times. Neutral lipid accumulation was visualized by Oil Red O (Abcam) staining as a functional marker for adipogenesis. Rinse cells with PBS 3 times for 10 min each.

    5. Image the stained CiDAs using epifluorescence microscopy, consisting of a Xenon lamp, an Axio Zoom V16 microscope, and Hamamatsu Flash 4.0 v3. Anticipate to observe over 50% of the cells is positive with the red color of Oil Red O staining (Figure 3C). The positive ratio of Oil Red O staining can be accessed using Image J, which is defined by the number of cells positive with red color divided by the total number of the cells.


  7. Inducing myogenic of CiDAs to test the multilineage potential of CiDAs

    1. Seed the harvested CiDAs in the wells of 6-well plates with a density of 3 × 105 cells per well, and culture them with MSCs expansion medium (CO2, 37 °C, and 95% humidity) until CiDAs reaching more than 90% confluence in the well.

    2. Exchange the expansion medium with myogenic medium (complete DMEM supplemented with 5% horse serum (HS) (Gibco), 0.1 μM dexamethasone (Sigma-Aldrich), and 50 μM hydrocortisone (Sigma-Aldrich)) for 10 days. Cycle the myogenic medium every 2 days.

    3. To test myogenesis efficiency after 10 days of incubation, fix CiDAs with 4% paraformaldehyde (Thermo Fisher Scientific) in PBS (Gibco). Wash the fixed sample three times with PBS. Incubate the fixed sample with PBS (Gibco) with 0.1% Triton X-100 (Sigma-Aldrich) for 30 min at 25 °C. Block nonspecific sites in the fixed sample using blocking buffer (PBS, 10% HS, and 0.1% Triton X-100) for an additional 60 min. Wash three times for 5 min each. Incubate the fixed cells with primary antibody anti-MyoD1 (Abcam) in blocking buffer. Rinse cells extensively in blocking buffer and incubate secondary antibody donkey anti-rabbit Alexa 488 (Thermo Fisher Scientific) for 1 h. Rinse cells by PBS 3 times for 10 min each. Incubate cells with DRAQ5 solution for 10 min before imaging.

    4. Image the stained CiDAs using confocal microscopy, with LAS X. Anticipate to observe over 50% of the cells are positive with the anti-MyoD1 staining (Figure 3D). The positive ratio of MyoD1 staining can be accessed using ImageJ, which is defined by the number of cells positive with anti-MyoD1 divided by the total number of the cells.


  8. Inducing myofibrogenesis of CiDAs to test the multilineage potential of CiDAs

    1. Seed the harvested CiDAs in the wells of 6-well plates with a density of 3 × 105 cells per well, and culture them with MSCs expansion medium (5% CO2, 37 °C, and 95% humidity) until CiDAs reach more than 70% confluence in the well.

    2. Exchange the expansion medium with myofibrogenic medium (complete DMEM supplemented with 2 ng/ml TGF-β (Abcam)) for 7 days. Cycle the myogenic medium every 2 days.

    3. To test myofibrogenesis efficiency after incubation, fix CiDAs with 4% paraformaldehyde (Thermo Fisher Scientific) in PBS (Gibco). Wash the fixed sample three times with PBS. Incubate the fixed sample with PBS (Gibco) with 0.1% Triton X-100 (Sigma-Aldrich) for 30 min at 25 °C. Block nonspecific sites in the fixed sample using blocking buffer (PBS, 10% HS, and 0.1% Triton X-100) for an additional 60 min. Wash three times for 5 min each. Incubate the fixed cells with primary antibody anti-α-SMA1 (Abcam) in blocking buffer. Rinse cells extensively in blocking buffer and incubate secondary antibody donkey anti-rabbit Alexa 488 (Thermo Fisher Scientific) for 1 h. Rinse cells by PBS 3 times for 10 min each. Incubate cells with DRAQ5 solution for 10 min before imaging.

    4. Image the stained CiDAs using confocal microscopy, with LAS X. Anticipate to observe over 50% of the cells are positive with the anti-α-SMA1 staining (Figure 3E). The positive ratio of α-SMA1 staining can be accessed using ImageJ, which is defined by the number of cells positive with anti-α-SMA1 divided by the total number of the cells.



      Figure 3. Expected results of multilineage induction from CiDAs. A. Expected ALP staining to test osteogenesis of CiDAs, at starting points, negative outcome, and positive outcome. Scale bar, 100 μm. (Step E). B. Expected calcium deposition staining to test osteogenesis of CiDAs, at starting points, negative outcome, and positive outcome. Scale bar, 50 μm. (Step E). C. Expected Oil Red O staining to test adipogenesis of CiDAs, at starting points, negative outcome, and positive outcome. Scale bar, 100 μm. (Step F). D. Expected MyoD staining to test myogenesis of CiDAs, at starting points, negative outcome, and positive outcome. Scale bar, 50 μm. (Step G). E. Expected α-SMA staining to test myofibrogenesis of CiDAs, at starting points, negative outcome, and positive outcome. Scale bar, 50 μm. (Step H)


    Trouble-shooting

  1. Low efficiency of adipogenesis of mesenchymal stem cells or adipose progenitors

    Possible cause: Low cell density before induction of adipogenesis.

    Possible repair: Increasing expansion time of MSCs/adipocyte progenitors culturing before exchanging to adipogenesis induction medium. MSCs/adipocyte progenitors should reach 90% confluency before exchanging to the adipogenesis induction medium.

  2. Contamination with non-adipocytes after ceiling culturing

    Possible cause: Insufficient digestion of adipocytes before density sorting.

    Possible repair: Increasing digestion time to trypsin the cells, and gently pipette the cells to sufficiently break down cell-to-cell contact and generate monodispersed cells.

  3. A limited number of adherent adipocytes after ceiling culturing

    Possible cause: Flow shear or harsh pipetting damages adipocytes during preparation.

    Possible repair: Be gentle when digest and pipette the cells; keep the cells at 4 °C during density sorting; avoiding flow shearing during fulfilling the culture flask, and be sure to remove all the air bubbles before turning the flask upside down.

  4. Adipocytes dying and detaching during culturing under high osmotic compression

    Possible cause: Quick exchange of hypertonic medium and quick osmotic shock.

    Possible repair: Exchange only half of the medium when changing medium, let the hypertonic medium slowly diffuse into the remaining culture medium, and reach the final concentration.


Future direction

There is significant heterogeneity in primary adipose tissue, which makes adipocytes isolated from different original sites behave much differently from each other. Thus, future works could involve optimizing the protocol to generate CiDAs from adipocytes of different origins. Secondly, mesenchymal stem cells or progenitor cells from different tissue usually exhibit different lineage potentials. Thus, for CiDAs generated from different origins of adipocytes, we also need to test their lineage potentials, which will help to define their practical applications in stem cell therapy, tissue engineering, and regenerative medicine. Another direction is further improving the efficiency of generating CiDAs and shortening the time required for the dedifferentiation of adipocytes. This could be done by combining mechanical stimulations and biochemical treatment. Current work from our group revealed that Wnt/β-catenin signaling plays an important role in adipocytes differentiation (Li et al., 2020b), which is also supported by another work (Gustafson and Smith, 2010). Another study from Kuang’s group revealed that Notch activation drove adipocyte dedifferentiation (Bi et al., 2016). Based on these understandings, a dedifferentiation cocktail medium, that not only mimics native physical stresses but also contains growth factors regulating Wnt/β-catenin signaling or Notch signaling, could more efficiently induce dedifferentiation of adipocytes and generate CiDAs. Overall, we hope that further development of this method may make CiDAs more accessible for many groups, and more stable for applications in regenerative medicine.

Acknowledgments

The authors would like to acknowledge the support from National Cancer Institute grant no. 1U01CA202123, National institute of general medical sciences grant no. 1R01GM140108, and the Jeptha H. and Emily V. Wade Award at MIT. A.S.M., B.R.S., T.-Y.S., and D.J.M. are supported by the NIH 5R01 DE013033 and 2R01 DE013349.

Competing interests

The authors declare no conflict of interests.

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简介

[摘要]目前的方法,以获得间充质干细胞(MSC)包括采样,培养,和扩大主要由脂肪,骨髓,和脐带组织的MSCs。然而,缺点是在总细胞在MSC池,和它们的衰减干性的数量有限在维生素- [R Ò扩张。作为替代资源,最近的天花板培养方法允许从成熟的脂肪细胞中生成去分化的脂肪细胞(DFAT)。然而,这种成熟脂肪细胞自发去分化的过程既费力又费时。本文描述了一种用于经修改协议在体外通过采用附加的物理刺激,其中脂肪细胞去分化TA KES扩充所述干性相关的优点的Wnt /β-catenin信号。具体来说,该协议利用含聚乙二醇(PEG)的高渗介质引入细胞外物理刺激以获得更高的效率,并引入更简单的脂肪细胞去分化程序。


[背景]脂肪组织由于其丰度大且侵袭性相对较低,因此是间充质干细胞(MSC)最具吸引力的来源之一(Shen等,2011 ;González-Cruz等,2012; Konno等人,2013)。脂肪来源的MSC,即从皮下脂肪组织的基质血管级分中分离,已被证实同时显示多谱系潜能的体外和体内(Anghileri等人,2008;冈萨雷斯。等人,2009;冈萨雷斯-雷伊等等人,2010; Jumabay等人,2010; Mao等人,2017和2019 ;Darnell等人,2018 )。要分离脂肪来源的MSC,广泛使用的方法是从脂肪组织中分离基质血管部分,然后通过荧光激活细胞分选(FACS)或培养物选择对MSC进行分类(Aronowitz等, 2015;Raposio等人,2017 ;Gentile等人,2019)。但是,脂肪组织的基质血管部分含有异质细胞群,而有限的细胞标志物可用于MSC的选择。这些使纯化来自脂肪的MSC变得困难(Gimble等,2011;González-Cruz等,2012; Konno等,2013)。

或者,脂肪细胞,而不是脂肪组织中的其他类型的细胞,可以在体外培养过程中自发分化为多能性间充质细胞,称为去分化脂肪(DFAT)细胞(Sugihara等人,1986;Shen等人,2011;Neugenes ,2011)。Taniguchi等人,2016年)。由于DFAT细胞的多能性和大量成熟脂肪细胞的存在,DFAT细胞已被认为是人出生后间充质干细胞的理想来源(Matsumoto等,2008; Shen等,2011 ;Daniel等,2011)。 Côtéet al。,20 19 )。然而,目前的顶棚培养脂肪细胞分化需要较长的持续时间(一般为4周),以使脂肪细胞SPON taneous LY失去所有明显梨皮d液滴(莱萨德等人,2015;谷口。等人,2016) 。因此,进一步增加了脂肪细胞分化的效率,缩短其处理时间是对于其更广泛的应用有吸引力。

脂肪细胞和脂肪祖细胞也是肿瘤微环境中的重要组成部分(Chandler等,2012; Seo等,201 5 ;Ling等,2020 )。最近的研究表明,脂肪细胞的去分化发生在肿瘤发展期间,这可能归因于活化的Wnt信号传导(Gustafson和Smith,2010 ;Bochet等人,2013)和Notch信号传导(Bi等人,2016)。再分的研究还表明,脂肪细胞c中的去分化乌尔德发生在体内小鼠模型小号(Bochet等人,2013;廖。等人,2015;王等人,2018) 。肿瘤的进展也极大地改变了局部物理微环境,包括升高的渗透压,增加的压缩力和基质硬化(Nia等人,2020)。这些物理提示在很大程度上影响脂肪基质细胞和癌细胞的细胞命运(Guo等,2017; Li等,2019和2020a ; Han等,2020)。确实,我们最近的研究报道了在体外模拟体内肿瘤中渗透压升高的渗透压的产生也可以诱导脂肪细胞的去分化(Li等人,2020b)。一致地,另一项研究还报告说,体内坚韧的植入物推动了周围周围脂肪细胞的去分化(Ma et al。,2019)。因此,这些研究启发我们开发了一种替代方案,通过机械去分化脂肪细胞来产生多能性间充质细胞。

这里描述的协议包括使用我们的高渗去分化培养基诱导和验证脂肪细胞重编程为多能间充质细胞的实验装置。我们还包括从前脂肪细胞或间充质干细胞生成脂肪细胞的程序,以及分化试验以确认CiDAs的多谱系潜力。

关键字:间充质干细胞, 去分化, 脂肪细胞, 压缩, Wnt /β连环蛋白信号

材料和试剂
试剂种类
1.最低基本培养基Eagle Alpha修改培养基(       Sigma-Aldrich,目录号:M8042)

2 胎牛血清(Gibco,目录号:10-082-147)

3. P enicillin /链霉素(GIBCO,目录号:15140148)     
4. P olyethylene乙二醇300(Sigma-Aldrich公司,目录号:8.07484)     
5.淘汰血清替代品(Gibco,目录号:10828-028)     
6.前脂肪细胞生长培养基2(Lonza,目录号:PT-8202)     
7. SingleQuots (Lonza,目录号:PT-9502)     
8. P araformaldehyde(VWR,目录号:IC0219998380)     
9. PBS(Sigma-Aldrich,目录号:P5119)     
10. Triton-X-100(Sigma-Aldric,目录号:X100) 
11.油红O(西格玛奥德里奇,目录号:O0625) 
12. DMEM(Sigma-Aldrich,品牌,目录号:D5546) 
13.高血清(Gibco,目录号:26050070) 
14. D哌替他松(Sigma-Aldrich,目录号:D4902) 
15. ħ ydrocortisone(Sigma-Aldrich公司,目录号:H0888) 
16. ħ ydrogen过氧化物(Sigma-Aldrich公司,目录号:H1009) 
17.甲NTI-MYOD1(Abcam公司,目录号:ab16148) 
18.驴抗兔Alexa 488(Invitrogen,目录号:R37118) 
19. β甘油磷酸(Sigma-Aldrich公司,目录号:G9422) 
20. L-抗坏血酸(Sigma-Aldrich,品牌,目录号:A4403) 
21. ELF -97(Invitrogen,目录号:E6588) 
22. TGF-β(R&d小号ystems,目录号:240-B) 
23.甲nti-的αSMA (Abcam公司,目录号:ab5694) 
24. DAPI(Thermo Scientific,品牌,目录号:62248) 
25.胰蛋白酶(2.5%)(Thermo Fisher Scientific,Gibco TM ,目录号:15090046) 

细胞培养塑料制品
T75和/或T25烧瓶(Corning,目录号:T75为430641U,T25为3056)
离心管(15 ml; 50 ml,Corning,目录号:430790; 430828)
冷冻小瓶(STARLAB,目录号:E3110-6122)
移液器吸头(TipOne ,STARLAB,目录号:S1111-3700; S1111-1706; S1111-6701)
35毫米细胞培养皿(Thermo Fisher Scientific,目录号:153066)
6孔板(Corning,隼® ,目录号:353934)
100 mm细胞培养皿(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:150464)

设备


1.离心机(Eppendorf,型号:5810)     
2. Bright-Line TM血细胞计数器(Sigma-Aldric,目录号:Z359629)                   
3.水浴(Thermo Scientific,目录号:TSCIR19)     
4. 37°C,5%CO 2的加湿培养箱(Thermo Fisher Scientific,贺利氏,型号:Heracell TM 150)     
5.徕卡TCS SP8共焦显微镜(徕卡)     
6.蔡司Axio Zoom V16显微镜(蔡司)     
7.氙弧灯(蔡司)     
8.滨松Orca Flash 4.0 V3(Scientifica )     
9.吸气(d RY真空PUM P /压缩机,韦尔奇真空-加德纳丹佛,型号:2511)     

软件


ImageJ(https://imagej.nih.gov/ij/)
LAS X(Leica Microsystems,曼海姆,德国)
HCImage (http://www.hamamatsu.com/)

程序


细胞文化性ë (图1,步骤1)
从美国典型细胞培养(ATCC)购买克隆衍生的小鼠MSC(OP9)。
展开的MSC(OP9)subconfluently在极限必需培养基Eagleα变介质补充有20%胎牛血清和1%青霉素/链霉素(完全在5%CO的条件MEM)2 ,37 ℃下和95%的湿度。
使用钙黄绿素乙酰氧基甲基和乙锭均二聚体1(Invitrogen,Eugene或OR)或锥虫蓝排除法(Beckman Coulter)评估细胞活力。
从Lonza购买皮下原代人前脂肪细胞。
在培养的原代人前脂肪细胞亚汇合在5%CO的条件中前脂肪细胞生长培养基-2(Lonza)中2 ,37 遵循制造商的说明,摄氏95度和95%湿度。

从前脂肪细胞或脂肪细胞的生成间充质干Ç厄尔(图1,步骤2)
诱导OP9的脂肪生成mMSCs中通过用MEM(Gibco)中含有15%的补充细胞淘汰赛硒朗姆酒更换(Gibco)中。
通过按照制造商的说明,在补充有胰岛素,地塞米松,消炎痛和异丁基甲基黄嘌呤的SingleQuots (Lonza)的前脂肪细胞生长培养基2(Lonza)中培养细胞,诱导人前脂肪细胞的脂肪形成。

从混合细胞群中筛选脂肪细胞
诱导脂肪形成后,胰蛋白酶消化混合细胞群。
将细胞悬浮液转移到15 ml的离心管中,并以低速离心(150 × g ,5分钟)。分化的脂肪细胞是漂浮在离心管中培养基的顶层上的,这是因为它们的密度比培养基低(图1,步骤3)。
占用ONL从在离心管的顶层,以及种子10层y中的分化的脂肪细胞5每培养瓶中的细胞(;25厘米猎鹰3012 2 )。将细胞在37 °C的培养基中孵育,该培养基由添加了20%胎牛血清的最低必需培养基Eagle Alpha修饰培养基组成。
用培养基完全充满烧瓶,为混合细胞提供无空气的环境(图1,第4步)。
在培养的第一天,将烧瓶倒置,使脂肪细胞在培养基中漂浮并粘附在烧瓶的顶部内表面(天花板表面)(图1,步骤5)。
细胞完全附着后将烧瓶倒回(大多数情况下1天就足够,不超过2天),以使细胞再次回到烧瓶底部(图1,第6步)。
要获得纯化和单分散的脂肪细胞群体,请充分消化并用移液管吸取。
轻轻洗去介质和所述残余未附着的细胞。
用5M培养脂肪细胞升介质包含在一个烧瓶在5%CO的条件2 ,37 ℃下和95%的湿度。也有一些麻木的undifferentiat ERS ED间充质干连接到烧瓶的顶面的细胞或前脂肪细胞。没有所述的介质的补充,这些细胞然后直接暴露于空气和不久死亡(图1,步骤7) 。



图1.产生压缩诱导的脂肪细胞去分化过程的示意图。1.将间充质干细胞或脂肪细胞祖细胞均匀接种在组织培养板中(步骤A)。2.使用脂肪生成培养基诱导MSC或脂肪细胞祖细胞的脂肪生成(步骤B)。3.用胰蛋白酶消化细胞,然后按密度对生成的脂肪细胞进行分类(步骤C1-C2)。4.使用细胞培养基和漂浮的脂肪细胞填充细胞培养瓶(步骤C3-C4)。5.翻转细胞培养瓶,使之通过吸顶培养使脂肪细胞附着在培养瓶底部(步骤C5)。6.将脂肪细胞完全附着在烧瓶底部后,翻转细胞培养瓶(步骤C6)。7.使用渗透压诱导脂肪细胞去分化(步骤D)。


诱导压缩诱导的脂肪细胞去分化
向烧瓶中抽吸并除去一半的培养基(2.5 ml)。
向烧瓶中加入2.5 ml高渗去分化培养基[补充了20%胎牛血清,1%青霉素/链霉素和4%PEG-300(分子量:300,v / v比率)的最低基本培养基Eagle Alpha修饰培养基] 。避免摇动烧瓶,并让高渗介质缓慢扩散并与烧瓶中的残留培养液混合。培养中的细胞在5%CO的条件2 ,37 ℃下和95%的湿度。
每三天更换一次高渗去分化培养基。渴望和除去4米升从烧瓶内的培养基中,再填充用4ml高渗脱分化培养基[极限必需培养基Eagleα变介质补充有20%胎牛血清,1%青霉素/链霉菌霉素和2%PEG-300 (MW:300,v / v比)] 。
避免完全去除培养基,并避免对脂肪细胞施加任何剪切力。由于脂肪细胞及其包含的脂滴s的脆弱特性,任何剪切力或快速的渗透压变化都可能损害细胞。
在培养过程中对培养的脂肪细胞成像。在10天之内,我们可能会观察到一半的脂肪细胞正在向间充质基质细胞样细胞迁移和去分化(图2)。其他脂肪细胞保留着较大的脂质滴,其中任何一个都不会进行去分化。



图2.压缩治疗9天之前和之后的脂肪细胞去分化。A.在渗透压诱导的去分化之前具有细胞脂质滴的脂肪细胞。乙。CiDAs的预期结果。红色箭头表示CiDAs所在的区域,而其他一些脂肪细胞则保留了其脂质小滴。比例尺100μ米。(步骤D)


用胰蛋白酶消化所有细胞,然后将它们转移到15 ml的离心管中。以150 × g的速度离心细胞5分钟。
除去顶部层残差adipocyt ES和超级游泳。重悬CiDAs中的MSC (补充有20%胎牛血清的极限必需培养基Eagleα变介质,1%青霉素/链霉素)的膨胀介质和种子细胞备份吨○烧瓶10层的密度5每培养瓶的细胞。培养中的细胞在5%CO的条件2 ,37 ℃下和95%的湿度。
每隔一天更换一次膨胀培养基,直到2周,直到CiDAs达到烧瓶表面的80%汇合为止。

Inducin的克成骨CiDAs测试的多向潜在CiDAs
种子收获CiDAs在6孔板的各孔与3个的密度× 10 5细胞每孔,并与培养他们的MSC扩增培养基(5%CO 2 ,37 ℃下和95%湿度),直到CiDAs达到更超过90%的井汇合度。
用成骨培养基(完整的DMEM补充10 mMβ-甘油磷酸和250 µM L-抗坏血酸)交换膨胀培养基。每两天循环一次成骨培养基。
为了测试ALP活性,修复CiDAs后6天培养中的成骨培养基中。Triton X-100可透化CiDA 。按照制造商的说明,用ELF -97(Thermo Fisher Scientific)染色固定的CiDA 。
图像的染色CiDAs使用外延荧光显微镜,由氙灯,的Axio上缩放V16显微镜,和滨松闪存4.0 V3。预期观察到超过50%的细胞呈阳性,ALP染色呈蓝色(图3A)。钙沉积的正比率可使用ImageJ,这是由所定义的访问NUM像素正与红色用图像的像素的总数除以BER。
要测试CiDAs成骨作用的矿物质沉积,首先从孔中吸出培养基。然后,将细胞在室温下于冰冷的70%乙醇中固定5分钟。抽吸酒精并用去离子水冲洗细胞两次(每次5分钟)。吸水并加入1 ml 2%茜素红S溶液,用氢氧化铵将其pH值调节至4.1-4.3。在室温下孵育孔板3分钟。吸出茜素红S溶液,并用2 ml水冲洗孔五次。
图像的染色CiDAs使用外延荧光显微镜,由氙灯,的Axio上缩放V16显微镜,和滨松闪存4.0 V3。预期观察到超过50%的表面呈阳性,且钙沉积染色呈红色(图3 B )。

诱导CiDAs的脂肪生成以测试CiDAs的多谱系潜力
种子收获CiDAs在我们LLS 6孔板与3个的密度× 10 5细胞每孔,并与MSC扩增培养基培养他们(5%CO 2 ,37 ℃下和95%湿度),直到CiDAs到达超过90%的孔汇合。
如果CiDAs是来自小鼠的MSC(OP9),原产È交换网与膨胀介质成脂(含有15%MEM(Gibco)中介质的KnockOut血清替代品(Gibco)中)。每两天循环一次成骨培养基。
如果CiDAs是从人原代前脂肪细胞始发,ê交换网用膨胀介质脂肪生成(前脂肪细胞生长培养基-2培养基补充有(Lonza)中SingleQuots (Lonza)中自由胰岛素,地塞米松,消炎痛和异丁基甲基黄嘌呤的)。每两天循环一次成骨培养基。
到试验后温育10天的脂滴积累,修复CiDAs用4%多聚甲醛(赛默飞世尔科技)在磷酸盐缓冲盐水(PBS)(Gibco)中的0.1%Triton X-100(Sigma-Aldrich公司)在25 30分钟℃ 。用PBS洗涤CiDAs 3次。通过油红O(Abcam)染色将中性脂质蓄积可视化,作为脂肪形成的功能标记。用PBS冲洗细胞3次,每次10分钟。
图像的染色CiDAs使用外延荧光显微镜,由氙灯,的Axio上缩放V16显微镜,和滨松闪存4.0 V3。预期观察到超过50%的细胞呈阳性,油红色O染色呈红色(图3C)。油红O染色的阳性率可以使用Image J,它是由限定访问NUM细胞的BER正用红色通过细胞的总数除以。

的诱导生肌CiDAs测试米的ultilineage潜在CiDAs
种子收获CiDAs在6孔板的各孔与3个的密度× 10 5细胞每孔,并与MSC扩增培养基培养它们(CO 2 ,37 ℃下和95%湿度),直到CiDAs达到超过90井中汇合百分比。
用肌原性培养基(完整的DMEM补充5%马血清(HS)(Gibco),0.1μM地塞米松(Sigma-Aldrich)和50μM氢化可的松(Sigma-Aldrich))交换扩增培养基10天。每2天循环一次肌原性培养基。
要在孵育10天后测试肌生成效率,请在PBS(Gibco)中用4%多聚甲醛(Thermo Fisher Scientific)固定CiDAs 。用PBS清洗固定的样品3次。在cubate固定的样品用PBS(Gibco)中的0.1%Triton X-100(Sigma-Aldrich公司)处理30分钟,在25 ℃下。使用封闭缓冲液(PBS,10%HS和0.1%Triton X-100)封闭固定样品中的非特异性位点,持续60分钟。洗3次,每次5分钟。将固定细胞与一抗MyoD1(Abcam)抗体在封闭缓冲液中孵育。在封闭缓冲液中充分漂洗细胞,然后将大肠杆菌抗体兔抗兔Alexa 488(Thermo Fisher Scientific)孵育1小时。用PBS冲洗细胞3次,每次10分钟。成像前,将细胞与DRAQ5溶液孵育10分钟。
图像的染色CiDAs使用共焦microsc OPY,与LAS X.预见到观察在50%的细胞是阳性与抗MYOD1染色(图3D)。MYOD1染色的阳性率可以使用ImageJ,这是由所定义的访问NUM细胞阳性的BER抗MYOD1除以细胞的总数。

诱导myofibrogenesis的CID作为测试的潜能研究CiDAs
种子收获CiDAs在6孔板的各孔与3个的密度× 10 5细胞每孔,并与MSC扩增培养基培养他们(5%CO 2 ,37 ℃下和95%湿度),直到CiDAs达到更超过井中70%的汇合度。
将扩张培养基与成肌纤维培养基(补充2 ng / ml lTGF-β(Abcam)的完全DMEM)交换7天。每2天循环一次肌原性培养基。
要在孵育后测试肌纤维形成效率,请在PBS(Gibco)中用4%多聚甲醛(Thermo Fisher Scientific)固定CiDAs 。用PBS清洗固定的样品3次。在cubate固定的样品用PBS(Gibco)中的0.1%Triton X-100(Sigma-Aldrich公司)处理30分钟,在25 ℃下。再使用封闭缓冲液(PBS,10%HS和0.1%Triton X-100)封闭固定样品中的非特异性位点,持续60分钟。每次清洗3分钟,每次5分钟。将固定细胞与第一抗体抗α-SMA1(Abcam)在封闭缓冲液中孵育。在封闭缓冲液中大量冲洗细胞,并将二抗驴抗兔Alexa 488(Thermo Fisher Scientific)孵育1小时。用PBS冲洗细胞3次,每次10分钟。成像前,将细胞与DRAQ5溶液孵育10分钟。
图像的染色CiDAs使用共nfocal显微镜,与LAS X.预见到观察在50%的细胞是阳性与抗α-SMA1染色(图3E)。α-SMA1染色的阳性率可以使用ImageJ,这是由所定义的访问NUM阳性细胞用抗α-SMA1的误码率除以细胞的总数。



图3. CiDAs多谱系诱导的预期结果。一。预期的ALP染色可在起点,阴性结果和阳性结果中测试CiDAs的成骨性。比例尺,100 μ米。(步骤E)。乙。预期的钙沉积染色可在起点,阴性结果和阳性结果中测试CiDAs的成骨性。比例尺,50 μ米。(步骤E)。Ç 。预期的油红O染色可在起点,阴性结果和阳性结果中测试CiDAs的脂肪生成。比例尺,100 μ米。(步骤F)。d 。预期的MyoD染色可在起点,阴性结果和阳性结果中测试CiDAs的发生。比例尺,50 μ米。(步骤G)。Ë 。预计α-SMA染色测试myofibrogenesis的CiDAs ,在开始点,负面的结果,并取得积极成果。比例尺,50 μ米。(步骤H)


故障排除

间充质干细胞或脂肪祖细胞成脂效率低
可能的原因:大号流脂肪生成诱导前的细胞密度。

可能的修复:在更换成脂诱导培养基之前,培养的MSCs /脂肪细胞祖细胞的扩增时间延长。MSC S /脂肪细胞祖细胞交换之前要达到90%汇合的脂肪生成诱导培养基。

吸顶培养后被非脂肪细胞污染
可能的原因:密度分选之前,我的脂肪细胞消化不足。

可能的修复:我D.加强消化时间与胰蛋白酶的细胞,并轻轻吸取细胞充分分解细胞与细胞的接触,并产生单分散细胞。

上限培养后有限数量的粘附脂肪细胞
可能的原因:˚F制备过程中的低剪切或苛刻的移液损害脂肪细胞。

可能的修复:乙消化,当E温柔吸管的细胞; 在密度分选过程中将细胞保持在4 °C ;避免在装满培养瓶期间出现剪切流,并确保在将培养瓶上下颠倒之前除去所有气泡。

在高渗透压下培养过程中脂肪细胞死亡和脱落
可能的原因:Q uick高渗中,快速渗透冲击的交流。

可能的修复:é Xchange的只有改变媒体时一半的媒体,让媒体高渗慢慢扩散到其余的培养基,并达到最终的浓度。


未来方向

在原发性脂肪组织中存在明显的异质性,这使得从不同的原始位点分离出的脂肪细胞的行为彼此差异很大。因此,未来的工作可能涉及优化协议以从不同来源的脂肪细胞生成CiDA 。其次,来自不同组织的间充质干细胞或祖细胞通常表现出不同的谱系潜能。因此,对于从不同来源的脂肪细胞生成的CiDA ,我们还需要测试其谱系潜力,这将有助于确定其在干细胞治疗,组织工程和再生医学中的实际应用。另一方向进一步提高生成的效率CiDAs并缩短所需的时间的脂肪细胞去分化。这可以通过结合机械刺激和生化处理来完成。我们小组目前的研究表明,Wnt /β-catenin信号在脂肪细胞分化中起着重要作用(Li等,2020b),这也得到了另一项研究的支持(Gustafson和Smith,2010)。Kuang研究小组的另一项研究表明,Notch激活导致脂肪细胞去分化(Bi等,2016)。基于这些理解,去分化混合物培养基不仅模拟天然的物理应激,而且还包含调节Wnt /β-catenin信号传导或Notch信号传导的生长因子,可以更有效地诱导脂肪细胞去分化并生成CiDAs 。总的来说,我们希望这种方法的进一步发展可以使CiDAs对许多人群更容易获得,并在再生医学中更稳定。


致谢


作者要感谢美国国家癌症研究所(National Cancer Institute)授予的No. 1U01CA202123 ,美国国立普通医学科学研究所 1R01GM140108,以及麻省理工学院的Jeptha H.和Emily V. Wade奖。NIH 5R01 DE013033和2R01 DE013349支持ASM,BRS,T.-YS和D.JM。


利益争夺


作者宣称没有利益冲突。


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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Li, Y., Mao, A. S., Seo, B. R., Zhao, X., Gupta, S. K., Chen, M., Han, Y. L., Shih, T., Mooney, D. J. and Guo, M. (2021). Generation of the Compression-induced Dedifferentiated Adipocytes (CiDAs) Using Hypertonic Medium. Bio-protocol 11(4): e3920. DOI: 10.21769/BioProtoc.3920.
  2. Li, Y., Mao, A. S., Seo, B. R., Zhao, X., Gupta, S. K., Chen, M., Han, Y. L., Shih, T. Y., Mooney, D. J. and Guo, M. (2020). Compression-induced dedifferentiation of adipocytes promotes tumor progression. Sci Adv 6(4): eaax5611.
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