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下腰痛(LBP)是导致残疾最常见的原因之一，多达80%的成年人在一生中经历过下腰痛。由此产生的生产力损失和财产损失每年高达2000亿美元。导致下腰痛的原因有很多，其中椎间盘退变被认为是最主要的原因。椎间盘退变后，细胞功能改变，导致细胞基质合成和降解之间的不平衡。蛋白多糖合成减少，II型胶原向I型胶原过渡从而导致椎间盘无法抵抗脊柱的机械力而产生疼痛。目前，退变性椎间盘疾病的治疗依赖于疼痛管理、运动以及手术治疗，但效果都不理想。来自美国西奈医学中心的 Dmitriy Sheyn与Zulma Gazit教授团队利用人类诱导性多能干细胞在猪体内分化为脊索细胞从而改善椎间盘退变的方法，为我们治疗椎间盘退变提供了一条新思路。 Figure 1. Step 1 - Differentiation of iPSCs into primitive streak mesoderm (PSM) cells. (A) Schematic representation of Step 1: iPSCs were treated with GSK3i and tested daily for expression of markers. (B) Morphological changes during GSK3i treatment. (C) Gene expression analysis of PSM cells shows a rapid decline in the expression of pluripotency markers (Nanog, Oct4, and Sox2) and an increase in mesodermal markers (MIXL1, BR, and FoxF1) in GSK3i-treated cells when compared to DMSO-treated cells. Results were calibrated relative to iPSCs (Day 0). Values are expressed as means ± standard errors (bars); n=6, *p<0.005; **p<0.01; ***p<0.001; ****p<0.0001. (D) Immunofluorescence staining of PSM cells for pluripotent and mesodermal markers was performed daily. The highest expression of mesodermal markers (Br, Mixl1, and FoxF1) was observed on Day 3 in conjunction with decreased expression of pluripotent markers (Oct4 and Sox2). (E) Flow cytometry of PSM cells for mesodermal CXCR4 marker and endoderm marker Sox17(48), showing that the PSM cells are homogeneous population of cells that differentiates exclusively to mesoderm. 接着研究者们使用基于核转染技术的非病毒转染系统过表达了参与脊索发育的主要转录因子之一Brachyury，从而将PSM细胞变为多能性干细胞来源的脊索细胞祖细胞（iNC progenitors）。通过免疫荧光观察，研究者们发现，转染2天后，细胞中脊索细胞祖细胞标志蛋白相对表达达到最高峰并且这种现象在4天，6天时急速下降。因此研究者们得出结论，转染后的第二天是诱导其成为多能干细胞来源的脊索细胞祖细胞最好的时机（Figure 2）。 Figure 2. Step 2 – Differentiation of PSM cells into iNC progenitors. (A) Schematic representation of Step 2: PSM cells were transfected with Br-encoding plasmid and placed in 2D culture. (B) On Days 2, 4, and 6 gene expression analyses were performed. They showed a rapid reduction in notochordal markers in both groups, but an elevation in the transfected gene over time. Values are expressed as means ± standard errors (bars); n=6, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (C) Immunofluorescence staining was performed for the NC markers Br and Keratin 8 (green), BASP1 and Keratin 18 (red), and SHH and FoxA2 (pink). The analyses confirmed the highest expression of NC markers on Day 2. We concluded that after 2 days in vitro NC progenitors start to lose their phenotype, and consequently we used Day 2 cells for our further experiments. 接着，研究者们模拟髓核的环境培养，利用Tetronic1307-Fibrinogen（TF）水凝胶，将iNC与BM-MSCs按1:1的比例混合培养，然后利用免疫荧光分析相应的标志蛋白表达，发现在缺氧条件下3D培养8周后，构建物中Basp1、CD24、角蛋白8、CTFG和FOXF1阳性表达增高，表明成功诱导其分化为脊索细胞（Figure 3）。 Figure 3. Step 3 – Maturation of iNC progenitors into iNCs in an NP-like environment and a paracrine effect on BM-MSCs. (A) PSM-Br Day 2 cells were embedded in TF gel and cultured in 2% O2 and NP media for up to 8 weeks. Additionally, the iNCs were mixed with BM-MSCs and co-cultured in TF gels in order to test the paracrine effect of iNCs on BM-MSCs. As a control, BM-MSCs were cultured alone in the same settings. (B) Every 2 weeks, TF gels were extracted and tested for gene expression of notochordal markers (Brachyury, Noto, Keratin 19 and Keratin 18) using qRT-PCR. Results show retention of the NC phenotype of the PSM-Br cells once cultured in the NP-like environment. Results are presented as mean RQs calibrated to PSM cells (Day 0). Values are expressed as means ± standard errors (bars); n=6, *p<0.05; **p<0.01; ****p<0.0001. (C) Immunofluorescence staining was performed for NC markers (Br, Keratins 8, 18 and 19, Noto, Gal3, and BASP1), one of the MSC markers (CD90) and NP markers (FOXF1, CTGF and CD24) at the end point of the study (8 weeks). NP and MSC markers were expressed in BM-MSC-included constructs and less so in iNCs only constructs. 随后研究者们针对之前的研究现象提出了假设，即混合细胞群中脊索细胞标记物的表达升高可能是由于在模拟脊索环境中iNCs对BM-MSCs的旁分泌作用所致。还有一种可能是，在脊索细胞条件下，iNCs的存活率可能高于BM-MSCs，因此，iNCs的标记物升高。为此，研究者们使用慢病毒载体，GFP标记iPSC和BM-MSCs，然后采用相同的条件培养，接着用流式细胞检测细胞活性，发现两种细胞活性并没有明显差异，从而否定第二种假设（Figure 4）。接着，研究者们就第一种猜想进行了实验，通过基因表达分析发现处理后BM-MSCs中分泌的Keratin 8, Keratin 19, Sox9, Aggrecan 以及Pax1的表达都升高了，从而证明了iNCs对BM-MSCs的旁分泌作用有比较明显的影响（Figure 5）。 Figure 4. Survival of iNC + BM-MSC cocultures in TF constructs. iNC and BM-MSC were stained with DiD lipophilic dye and encapsulated in TF gels alone or with iNC-GFP or BM-MSC-GFP according to the diagram (A). The constructs were cultured in hypoxic NP conditions for 8 weeks, gels digested, the cells recovered and counted using Countess to assess viability (B, C). Flow cytometry plots show distribution of GFP expressing cells (green) or DiD-labeled cells (pink) (D). Quantitative analysis of separate constructs shows ratio between the green and pink cells (E) or when separated and analyzed based on GFP expressing cells (F) or DiD-labeled cells (G). Values are expressed as means ± standard errors (bars), n=6. Figure 5. iNC functional assay: paracrine effect on BM-MSC. Conditioned medium (CM) was collected from iNCs or primary porcine NCs (pNC) grown in alginate beads. BM-MSCs in alginate beads were grown in either iNC-derived conditioned medium (iNC-CM) or in porcine NC-derived conditioned media (pNC-CM) and analyzed on Day 7. Values are relative to BM-MSCs grown in regular media. Values are expressed as means ± standard errors (bars), n=5; *p<0.05; ***p<0.001; ****p<0.0001. Figure 6. Porcine IVD degenerated and injected with iNCs: imaging analysis. (A) Schematic diagram for the experimental design. (B) Three levels of IVDs were subjected to annular puncture with a 14G needle under fluoroscopic guidance. (C) The degeneration process was imaged using MRI. The first image was obtained before induction of degeneration and shows all healthy IVDs. Subsequent images were taken 4, 8, or 12 weeks after annular puncture. Yellow arrows indicate injured IVDs and white arrows indicate healthy IVDs. (D) 4 weeks after induction of degeneration, DiI-labeled iNCs, BM-MSCs, or hydrogel alone were injected into the NP. (E) qCEST imaging that was previously correlated to the pH measured inside the disc. Values are expressed as means ± standard errors (bars), n=3; ****p<0.0001 (comparing to hydrogel only). Figure 7. iNCs survive and maintain their phenotype 8 weeks after intradiscal injection. The IVDs were harvested 8 weeks after the cell injections and subjected to histological analysis and H&E staining. (A) Magnification of the NP area. (B) The injected iNCs were detected using immunofluorescence staining against Brachyury, Keratins 8, 18, and 19, and CTGF, and subjected to confocal microscopy. Colocalization of the notochordal markers and DiI-labeled iNCs or the DiO-labeled BM-MSCs in the NP area in vivo can be observed. (C) Gene expression analysis of the harvested IVDs at Week 12 after induction of degeneration and Week 8 after the cell/hydrogel injections shows expression of the human notochordal markers (Brachyury and Noto) in the NP and endplate areas, and expression of the NP differentiation markers GDF6 and Sox9 in the NP area. Values are expressed as means ± standard errors (bars) and are relative to the hydrogel only group; n=3; *p<0.05; ***p<0.001; ****p<0.000. NP – nucleus pulposus, AF – annulus fibrosus, EP – Endplate. 综上所述，研究者们成功发现人类诱导性多能干细胞可在猪体内分化为脊索细胞，从而减少椎间盘的退变。该研究为椎间盘退变的治疗指明了一条新思路。 本研究由来自美国西奈医学中心的 Dmitriy Sheyn与Zulma Gazit教授团队完成，并于2019年10月发表于Theranostics。 论文信息：Dmitriy Sheyn*, Shiran Ben-David, Wafa Tawackoli, Zhengwei Zhou, Khosrawdad Salehi, Maxim Bez, Sandra De Mel, Virginia Chan, Joseph Roth, Pablo Avalos, Joseph C. Giaconi, Haneen Yameen, Lena Hazanov, Dror Seliktar, Debiao Li, Dan Gazit, Zulma Gazit*. Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model. Theranostics 2019, 9: 7506-7524. |
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