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Mar 2021

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Skeletal Stem Cell Isolation from Cranial Suture Mesenchyme and Maintenance of Stemness in Culture
从颅缝间充质中分离骨骼干细胞和培养干细胞的维持   

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Abstract

Skeletal stem cells residing in the suture mesenchyme are responsible for calvarial development, homeostatic maintenance, and injury-induced repair. These naïve cells exhibit long-term self-renewal, clonal expansion, and multipotency. They possess osteogenic abilities to regenerate bones in a cell-autonomous manner and can directly replace the damaged skeleton. Therefore, the establishment of reliable isolation and culturing methods for skeletal stem cells capable of preserving their stemness promises to further explore their use in cell-based therapy. Our research team is the first to isolate and purify skeletal stem cells from the calvarial suture and demonstrate their potent ability to generate bone at a single-cell level. Here, we describe detailed protocols for suture stem cell (SuSC) isolation and stemness maintenance in culture. These methods are extremely valuable for advancing our knowledge base of skeletal stem cells in craniofacial development, congenital deformity, and tissue repair and regeneration.

Keywords: Bone regeneration (骨再生), Calvaria (头顶), Cell-based therapy (细胞疗法), Craniofacial (颅面), Skeletal stem cell (骨骼干细胞), Sphere culture (球形培养), Skeletogenic mesenchyme (成骨间充质), Suture stem cell (缝合干细胞), Mesenchymal stem cell (间充质干细胞), Osteogenesis (成骨)

Background

Conventional methods have shown successful isolation of mesenchymal stromal cells (MSCs) from bone marrow and other tissues (Friedenstein et al., 1974; da Silva Meirelles et al., 2006). MSCs contain skeletal stem cells (SSCs) with self-renewing and skeletogenic differentiating abilities (Sacchetti et al., 2007). However, only ~10–20% of them are genuine SSCs (Robey et al., 2014). The difficulties in engraftment, survival, and differentiation of the transplanted MSCs have also been well documented (Caplan and Correa, 2011; Zeitouni et al., 2012). Furthermore, the cellular source of the endogenous MSC remains unknown. Here, we describe a protocol for the isolation of SSCs from the calvarial suture mesenchyme. These SuSCs have been demonstrated to generate bone at a single cell level upon kidney capsule transplantation, which can faithfully assess SuSC properties in vivo (Maruyama et al., 2016). It is also important to develop a protocol capable of maintaining their stemness in vitro. The successful establishment of the skeletal stem cell culture protocol permits further assessments of stem cell characteristics, e.g., self-renewal, proliferation, fate determination, and differentiation in an ex vivo setting. The preservation of stem cell stemness in culture is also critical for bone tissue engineering and opens the possibility of exploring next-generation therapeutics. We also describe an ex vivo protocol to culture SuSCs for an extended period. The cultured SuSCs can generate bones upon implantation to the ectopic site (Maruyama et al., 2021). We demonstrate that this culture method can determine the label-retaining ability and asymmetric division of SuSCs. It also provides an outstanding system to further our examination of additional stem cell characteristics, e.g., cell fate determination, generation of skeletal progenitors, and skeletogenic differentiation, as well as studies at the transcriptome (RNA-seq), epigenetics (ATAC-seq and ChIP-seq), and single-cell levels (scRNA-seq). Using these new tools, we can reconstruct lineage relationships between cells within craniofacial and skeletal tissues—a long-standing challenge in biology. These are extremely important advancements in stem cell-based therapy for bone regeneration and repair.

Materials and Reagents

  1. Cell strainer 40 µm Nylon (Falcon, catalog number: 352340)

  2. Ultra-Low Attachment Surface 24-well plate (Corning, catalog number: 3473)

  3. Dulbecco’s Phosphate-Buffered Saline without calcium & magnesium (DPBS; Corning, catalog number: 21-031-CV), 2 years shelf life at room temperature

  4. 0.2% collagenase A (Roche, catalog number: 11088793001), freshly reconstituted from lyophilized stock in PBS

  5. 10 mM HEPES (1 M buffer solution; Gibco, catalog number: 15630-080), 24 months shelf-life at 4°C

  6. Penicillin-Streptomycin (Gibco, catalog number: 15140-122), 12 months shelf life at -20°C

  7. 100 µg/mL transferrin (Sigma, catalog number: T8158) at -20°C

  8. 20 nM progesterone (Sigma, catalog number: P8783) at -20°C

  9. 30 nM Sodium selenite (Sigma, catalog number: S8295) at -20°C

  10. 60 nM Putrescine dihydrochloride (Sigma, catalog number: P5780), 4 years shelf life at -20°C

  11. EGF (Mouse EGF Recombinant Protein; Gibco, catalog number: PMG8041), 1 year shelf life at -20°C

  12. bFGF (mouse FGF-basic Recombinant Protein; Gibco, catalog number: PMG0035), 1 year shelf life at -20°C

  13. B27 supplement (Gibco, catalog number: 17504044,), 1 year shelf life at -20°C

  14. Insulin (Sigma, catalog number: I1882) at -20°C

  15. 0.25% trypsin (Gibco, catalog number: 25200-056), 2 years shelf life at -20°C

  16. Soybean trypsin inhibitor (SBTI; Sigma, catalog number: T9128) at -20°C

  17. Digestion Buffer (see Recipes)

  18. Sphere Culture Media (see Recipes)

  19. 2× SBTI Solution (see Recipes)

Equipment

  1. Shaker (Thermo Scientific, MaxQtm 4000 Benchtop Orbital Shakers)

  2. Centrifuge (Thermo Scientific, Sorvall ST 16R Centrifuge)

  3. Incubator (Thermo Forma, Series II Water Jacketed CO2 Incubator)

Procedure

  1. Isolation of mesenchymal cells from suture mesenchyme

    1. Dissect a 2 mm wide portion of calvarial tissue containing the sagittal (SAG) suture and its adjacent parietal bones from mouse calvarium (Figure 1).



      Figure 1. Schematic of mouse calvarial bones and sutures.

      F, frontal bone; P, parietal bone; IP, interparietal bone.


    2. Hint: It is easy to remove the calvarium from the meninges. The cutout size should be as small as possible, to limit the amounts of contaminant cells from the bone marrow and periosteum.

    3. Grab both sides of the parietal bones with two forceps and gently pull them apart (Figure 2).



      Figure 2. Schematic diagrams illustrating the separation of bone pieces to release the cells from the suture mesenchyme.


    4. Attention: The dissected calvarial tissue containing two small parietal bone pieces must be separated in the midline to expose the suture mesenchyme. Otherwise, the number of skeletal stem cells would be significantly reduced.

    5. Hint: To avoid contamination of stem cells from the bone marrow, do not cut the separated calvarial tissue into pieces (this process is performed for calvarial cell isolation). Do not keep the dissected tissue on ice, rather quickly incubate it with Digestion Buffer as described in step 6.

    6. Submerge the separated pieces in 40 mL of Digestion Buffer and incubate in a shaker at 200 × g (MaxQtm 4000 Benchtop Orbital Shakers) and 37°C for 1 h.

    7. Filter the dissociated cells through a 40 µm strainer.

    8. Spindown the cells by centrifugation at 400 × g and 4°C for 7 min.

    9. Remove the supernatant to collect the cell pellets.

    10. Hint: About 5 × 104 cells can be obtained from one sagittal suture

    11. Hint: Suture mesenchymal cells have been successfully isolated from the calvaria of mice at postnatal day 10 (P10) and P28.


  2. Sphere culture

    1. Resuspend the cell pellets with Sphere Culture Media and count the cell number.

    2. Seed the cells in 2 mL of sphere culture media at 104–105 cells per well on an Ultra-Low Attachment Surface 24-well plate (Figure 3A).

    3. Carefully change half the amount of the culture media in the culture every other day, without disturbance of cell settling.

    4. Hint: Sphere formation will be visible within 10 to 14 days (Figure 3B and Maruyama et al., 2021).



      Figure 3. Representative images showing cells after 1 day (A) and 8 days (B) of the sphere culture. Scale bars, 200 µm.


    5. Hint: Most of the sphere is derived from a single stem cell (Maruyama et al., 2021).

    6. Hint: Stem cell stemness, e.g., bone-forming ability and quiescence, has been demonstrated to be maintained in culture using the 1st and 3rd passage of the sphere by kidney capsule transplantation and label-retaining analyses (Maruyama et al., 2021).

    7. Hint: Bone-forming ability is assessed by the kidney capsule transplantation assay, followed by von Kossa staining and immunostaining of osteoblast markers. Quiescence is examined by the pulse-chase assay in sphere culture.


  3. Passaging spheres

    1. Some spheres may be weakly attached to the bottom of the well. To detach the sphere, gently pipette the culture media up and down.

    2. The cell suspension is collected by centrifugation at 300 × g and 4°C for 7 min.

    3. Remove the supernatant and tap the tube to gently break up the pellets.

    4. Add 1 mL of 0.25% Trypsin to resuspend the pellet, followed by incubation at 37°C for 5 min.

    5. Add 1 mL of 2× SBTI solution to stop the activity of the trypsin.

    6. Centrifuge at 300 × g and 4°C for 7 min.

    7. Remove supernatant and tap the tube to gently break up the pellets.

    8. Resuspend the cells in the Sphere Culture Media and count cell numbers.

    9. Seed the cells at 104–105 cells per well.

    10. Hint: This culture method has been limited to five passages.

Data analysis

  1. Sphere measurement

    1. Gently pipette the medium to detach the spheres from the culture plate.

    2. Hint: No special pipet tips are required, only general ones that generate a little media flow.

    3. Take images using a camera under a microscope, e.g., NIKON CB-115 (Figures 5, 6, Supplemental Figures S9, S10 of Maruyama et al., 2021).

    4. Measure the sphere number and size by ImageJ (Figures 6, Supplemental Figures S9, S10 of Maruyama et al., 2021).

    5. Hint: For the basic function of ImageJ: “ImageJ Basics (PDF file)” on the official page (https://imagej.nih.gov/ij/index.html) under “Documentation”-“Tutorials and Examples”.

    6. Count spheres that are >20 µm in diameter.

    7. Perform at least three independent experiments for statistical evaluation.

    8. Examine the statistical significance of sphere number and size using a two-sided Student’s t-test.


  2. Evaluation of bone formation after kidney capsule transplantation

    1. Perform whole-mount von Kossa staining of bone in the transplanted kidney capsule (Figures 4, 5, 6, Supplemental Figures S9, S10 of Maruyama et al., 2021).

    2. Image using a microscope, e.g., NIKON CB-115.

    3. Measure the size of the stained area by ImageJ.

    4. Perform at least three independent experiments for statistical evaluation.

    5. Examine the statistical significance of sphere number and size using a two-sided Student’s t-test (Supplemental Figure S10 of Maruyama et al., 2021).

Recipes

  1. Digestion Buffer (40 mL)

    DPBS containing 0.2% collagenase A, 1% Penicillin-Streptomycin, and 10 mM HEPES

  2. Sphere Culture Media

    25 µg/mL insulin

    100 µg/mL transferrin

    20 nM progesterone

    30 nM Sodium selenite

    60 nM putrescine

    20 ng/mL EGF

    20 ng/ml bFGF

    1× B27 supplement

    1% Penicillin-Streptomycin

  3. 2× SBTI Solution

    2 mg SBTI in 1 mL of DPBS

Acknowledgments

The authors thank former and current lab members for their technical and intellectual support. This work is supported by the National Institutes of Health (DE15654, DE269369) and NYSTEM (C029558) to W.H. This protocol is derived from the original research paper published in Science Translational Medicine (Maruyama et al., 2021).

Competing interests

The authors declare no competing financial interests.

Ethics

Care and use of experimental animals described in this work comply with guidelines and policies of the University Committee on Animal Resources at the University of Rochester and IACUC at the Forsyth Institute.

References

  1. Caplan, A. I. and Correa, D. (2011). The MSC: an injury drugstore. Cell Stem Cell 9(1): 11-15.
  2. da Silva Meirelles, L., Chagastelles, P. C. and Nardi, N. B. (2006). Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci (11): 2204-2213.
  3. Friedenstein, A. J., Chailakhyan, R. K., Latsinik, N. V., Panasyuk, A. F. and Keiliss-Borok, I. V. (1974). Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 17(4): 331-340.
  4. Maruyama, T., Jeong, J., Sheu, T. J. and Hsu, W. (2016). Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration. Nat Commun 7: 10526.
  5. Maruyama, T., Stevens, R., Boka, A., DiRienzo, L., Chang, C., Yu, H. I., Nishimori, K., Morrison, C. and Hsu, W. (2021). BMPR1A maintains skeletal stem cell properties in craniofacial development and craniosynostosis. Sci Transl Med 13(583): eabb4416.
  6. Robey, P. G., Kuznetsov, S. A., Riminucci, M. and Bianco, P. (2014). Bone marrow stromal cell assays: in vitro and in vivo. Methods Mol Biol 1130: 279-293.
  7. Sacchetti, B., Funari, A., Michienzi, S., Di Cesare, S., Piersanti, S., Saggio, I., Tagliafico, E., Ferrari, S., Robey, P. G. and Riminucci, M. (2007). Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131(2): 324-336.
  8. Zeitouni, S., Krause, U., Clough, B. H., Halderman, H., Falster, A., Blalock, D. T., Chaput, C. D., Sampson, H. W. and Gregory, C. A. (2012). Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration. Sci Transl Med 4(132): 132ra155.

简介

[摘要]存在于缝线间充质中的骨骼干细胞负责颅骨发育、稳态维持和损伤诱导的修复。这些幼稚细胞表现出长期的自我更新、克隆扩增和多能性。它们具有以细胞自主方式再生骨骼的成骨能力,可以直接替换受损的骨骼。因此,建立能够保持其干性的骨骼干细胞的可靠分离和培养方法,有望进一步探索它们在基于细胞的治疗中的应用。我们的研究团队率先从颅骨缝中分离和纯化骨骼干细胞,并展示了它们在单细胞水平上产生骨骼的强大能力。在这里,我们描述了缝合干细胞 ( SuSC ) 分离和培养干细胞维持的详细协议。这些方法对于推进我们在颅面发育、先天性畸形以及组织修复和再生方面的骨骼干细胞知识库非常有价值。


[背景] 常规方法已显示成功地从骨髓和其他组织中分离间充质基质细胞 (MSC) (Friedenstein等人,1974;da Silva Meirelles等人,2006) 。 MSCs 含有具有自我更新和成骨分化能力的骨骼干细胞 (SSCs) (Sacchetti et al. , 2007) 。然而,其中只有约 10 – 20% 是真正的 SSC (Robey等人,2014 年) 。移植的 MSC 在植入、存活和分化方面的困难也得到了充分证明(Caplan 和 Correa,2011;Zeitouni等,2012) 。此外,内源性MSC的细胞来源仍然未知。在这里,我们描述了从颅骨缝合间充质中分离 SSC 的协议。这些SuSC已被证明在肾包膜移植后在单细胞水平上生成骨,这可以忠实地评估体内SuSC的特性(Maruyama等人,2016 年) 。开发一种能够在体外维持其干性的方案也很重要。骨骼干细胞培养方案的成功建立允许进一步评估干细胞特征,例如,自我更新、增殖、命运决定和离体环境中的分化。在培养中保存干细胞干细胞对骨组织工程也至关重要,并为探索下一代疗法开辟了可能性。我们还描述了一种用于长时间培养SuSC的体外协议。培养的SuSC可在植入异位部位后生成骨骼(Maruyama等人,2021) 。我们证明了这种培养方法可以确定SuSCs的标签保留能力和不对称分裂。它还提供了一个出色的系统来进一步检查其他干细胞特征,例如,细胞命运测定、骨骼祖细胞的产生和骨骼分化,以及转录组 (RNA-seq)、表观遗传学 (ATAC-seq) 的研究和ChIP -seq)和单细胞水平( scRNA -seq)。使用这些新工具,我们可以重建颅面和骨骼组织内细胞之间的谱系关系——这是生物学中长期存在的挑战。这些是基于干细胞的骨再生和修复疗法中极其重要的进步。

关键字:骨再生, 头顶, 细胞疗法, 颅面, 骨骼干细胞, 球形培养, 成骨间充质, 缝合干细胞, 间充质干细胞, 成骨

材料和试剂
1.细胞过滤器40 µm尼龙(Falcon,目录号:352340)
2.超低附着表面 24 孔板(Corning,目录号:3473)
3.不含钙和镁的 Dulbecco 磷酸盐缓冲盐水(DPBS;Corning,目录号:21-031-CV),室温下保质期为 2 年
4.0.2%胶原酶A(Roche,目录号:11088793001),从PBS中的冻干原液新鲜重构
5.°C 下24 个月的保质期
6.°C 下的 12 个月保质期
7.100 µg/mL 转铁蛋白(Sigma,目录号:T8158) ,-20 °C
8.20 nM黄体酮(Sigma,目录号:P8783),-20 °C
9.30 nM亚硒酸钠(Sigma,目录号:S8295),-20 °C
10.60 nM二盐酸腐胺(Sigma,目录号:P5780),-20 °C 4年保质期
11.°C 下的 1 年保质期
12.bFGF (小鼠 FGF 碱性重组蛋白;Gibco,目录号:PMG0035),-20 °C 下的 1 年保质期
13.°C 下的 1 年保质期
14.胰岛素(Sigma,目录号:I1882)在-20 °C
15.°C 2年保质期
16.大豆胰蛋白酶抑制剂(SBTI;Sigma,目录号:T9128)在-20 °C
17.消化缓冲液(见食谱)
18.球形培养基(见食谱)
19.2 × SBTI 解决方案(见配方)


设备


1.摇床(Thermo Scientific, MaxQtm 4000 台式轨道摇床)
2.离心机(Thermo Scientific, Sorvall ST 16R 离心机)
3.培养箱(Thermo Forma,系列 II 水套 CO 2培养箱)


程序


A.从缝合间充质中分离间充质细胞
1.解剖 2 毫米宽的颅骨组织,其中包含矢状 (SAG) 缝合线及其相邻的顶骨(图1 )。


 
图1.小鼠颅骨和缝合线示意图。 
F,额骨; P,顶骨; IP,顶骨。


2.提示:从脑膜中取出颅骨很容易。切口尺寸应尽可能小,以限制来自骨髓和骨膜的污染细胞的数量。
3.用两个镊子抓住顶骨的两侧,轻轻地将它们拉开(图 2)。


 
图 2. 示意图说明骨片分离以从缝合间充质中释放细胞。


4.注意:包含两个小顶骨片的解剖颅骨组织必须在中线分开,以暴露缝合间质。否则,骨骼干细胞的数量将显着减少。
5.提示:为避免从骨髓中污染干细胞,请勿将分离的颅骨组织切成碎片(此过程用于颅骨细胞分离)。不要将解剖的组织放在冰上,而是按照步骤 6 中的说明用消化缓冲液快速孵育。
6.将分离的片段浸入 40 mL 的消化缓冲液中,并在 200 × g ( MaxQtm 4000 台式轨道摇床)和 37°C 的摇床上孵育 1 小时。
7.通过 40 μm 过滤器过滤分离的细胞。
8.× g 和 4°C 下离心 7 分钟来离心细胞。
9.去除上清液以收集细胞沉淀。
10.提示:从一根矢状缝中可以获得大约 5 × 10 4 个细胞
11.提示:已在出生后第 10 天(P10)和 P28 从小鼠颅盖骨中成功分离缝合间充质细胞。


B.球体文化
1.用球形培养基重悬细胞沉淀并计算细胞数。
2.在 2 mL 的球体培养基中播种细胞,在超低附着表面 24 孔板上每孔10 4 – 10 5 个细胞(图 3A)。
3.每隔一天小心地更换培养物中一半的培养基, 不受细胞沉降的干扰。
4.提示:球体形成将在 10 到 14 天内可见(图 3B 和 Maruyama等人,2021)。


 
图 3. 显示球体培养 1 天 (A) 和 8 天 (B) 后的细胞的代表性图像。比例尺,200 µm。


5.提示:大部分球体源自单个干细胞(Maruyama等人,2021 年) 。
6.通过肾包膜移植和标签保留分析,已证明使用第 1 次和第 3 次球体传代可在培养中维持干细胞干细胞性,例如骨形成能力和静止(Maruyama等人,2021 年) .
7.肾包膜移植试验评估骨形成能力,然后进行 von Kossa染色和成骨细胞标志物的免疫染色。通过球培养中的脉冲追踪测定来检查静止状态。


C.传球球
1.一些球体可能很弱地附着在井底。要分离球体,请上下轻轻吸管培养基。
2.×离心收集细胞悬液 g和 4°C 7 分钟。
3.取出上清液并轻敲试管以轻轻打碎颗粒。
4.加入 1 mL 0.25% 胰蛋白酶重悬沉淀,然后在 37°C 下孵育 5 分钟。
5.添加 1 mL 的 2 × SBTI 溶液以停止胰蛋白酶的活性。
6.300 ×离心机 g和 4°C 7 分钟。
7.去除上清液并轻敲试管以轻轻打碎颗粒。
8.重悬球体培养基中的细胞并计数细胞数。
9.4 – 10 5 个细胞播种细胞。
10.提示:这种培养方法仅限于五个段落。


数据分析


A.球体测量
1.轻轻移液器将球体从培养板中分离出来。
2.提示:不需要特殊的移液器吸头,只需要产生少量培养基流量的通用吸头即可。
3.使用显微镜下的相机拍摄图像,例如NIKON CB-115 (图 5、6,Maruyama等人的补充图 S9、 S10,2021) 。
4.通过 ImageJ 测量球体数量和大小(Maruyama等人的图 6、补充图 S9、 S10,2021) 。
5.提示:ImageJ的基本功能:官方页面( https://imagej.nih.gov/ij/index.html)“Documentation”-“Tutorials and Examples”下的“ ImageJ Basics(PDF文件)”。
6.计算直径 > 20 µm 的球体。
7.执行至少三个独立实验进行统计评估。
8.使用双面学生t检验检查球体数量和大小的统计显着性。


B.肾包膜移植后骨形成的评估
1.对移植的肾囊中的骨骼进行整体 von Kossa染色(图 4、5、6,补充图 S9、Maruyama等人的S10,2021) 。
2.使用显微镜拍摄的图像,例如., NIKON CB-115。
3.通过 ImageJ 测量染色区域的大小。
4.执行至少三个独立实验进行统计评估。
5.使用双面学生t检验检查球体数量和大小的统计显着性(Maruyama等人的补充图S10,2021 ) 。


食谱


1.消化缓冲液 (40 mL)
DPBS 含有 0.2% 胶原酶 A、1% 青霉素-链霉素和 10 mM HEPES
2.球体文化传媒
25 µg/mL 胰岛素
100 µg/mL 转铁蛋白
20纳米黄体酮
30 nM 亚硒酸钠
60纳米腐胺
20 纳克/毫升表皮生长因子
20 纳克/毫升 bFGF
1 × B27 补充
1% 青霉素-链霉素
3.2 × SBTI 解决方案
1 mL DPBS 中的 2 mg SBTI


致谢


作者感谢前任和现任实验室成员的技术和智力支持。这项工作得到了美国国立卫生研究院 (DE15654、DE269369) 和 NYSTEM (C029558) 对 WH 的支持。该协议源自发表在《科学转化医学》上的原始研究论文 (丸山等人,2021) 。


利益争夺


作者声明没有竞争的经济利益。


伦理


这项工作中描述的实验动物的护理和使用符合罗切斯特大学动物资源大学委员会和福赛斯研究所 IACUC 的指导方针和政策。


参考


1.Caplan, AI 和 Correa, D. (2011)。 MSC:伤害药店。 细胞干细胞9(1):11-15。
2.da Silva Meirelles, L.、Chagastelles, PC 和 Nardi, NB (2006)。间充质干细胞几乎存在于所有产后器官和组织中。 J 细胞科学119(Pt 11):2204-2213。
3.Friedenstein, AJ, Chailakhyan, RK, Latsinik, NV, Panasyuk, AF 和 Keiliss-Borok, IV (1974)。负责转移造血组织微环境的基质细胞。体外克隆和体内再移植。 移植17(4):331-340。
4.Maruyama, T.、Jeong, J.、Sheu, TJ 和 Hsu, W. (2016)。缝合间充质干细胞在颅面骨发育、修复和再生中的作用。 国家通讯7:10526。
5.Maruyama, T.、Stevens, R.、Boka, A.、DiRienzo, L.、Chang, C.、Yu, HI、Nishimori, K.、Morrison, C. 和 Hsu, W. (2021)。 BMPR1A 在颅面发育和颅缝早闭中维持骨骼干细胞特性。 Sci Transl Med 13(583):eabb4416。
6.Robey, PG, Kuznetsov, SA, Riminucci, M. 和 Bianco, P. (2014)。骨髓基质细胞测定:体外和体内。 方法 Mol Biol 1130:279-293。
7.Sacchetti, B., Funari, A., Michienzi, S., Di Cesare, S., Piersanti, S., Saggio, I., Tagliafico, E., Ferrari, S., Robey, PG, Riminucci, M.,等人。 (2007 年)。骨髓血窦中的自我更新骨祖细胞可以组织造血微环境。 单元格131(2):324-336。
8.Zeitouni, S., Krause, U., Clough, BH, Halderman, H., Falster, A., Blalock, DT, Chaput, CD, Sampson, HW 和 Gregory, CA (2012)。用于增强骨再生的人间充质干细胞衍生基质。 Sci Transl Med 4(132):132ra155。


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Copyright: © 2022 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. Maruyama, T., Yu, H. I. and Hsu, W. (2022). Skeletal Stem Cell Isolation from Cranial Suture Mesenchyme and Maintenance of Stemness in Culture. Bio-protocol 12(5): e4339. DOI: 10.21769/BioProtoc.4339.
  2. Maruyama, T., Stevens, R., Boka, A., DiRienzo, L., Chang, C., Yu, H. I., Nishimori, K., Morrison, C. and Hsu, W. (2021). BMPR1A maintains skeletal stem cell properties in craniofacial development and craniosynostosis. Sci Transl Med 13(583): eabb4416.
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