Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Science, Fukuoka, Japan.
PLoS One. 2013 May 7;8(5):e62506. doi: 10.1371/journal.pone.0062506. Print 2013.
Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker(+) cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes.
METHODOLOGY/PRINCIPAL FINDINGS: Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγ(null) mice. GFP(+) cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP(+) donor-derived cells, GFP(+)CFP(+) fused cells, and CFP(+) recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin(-/low)CD45(+) hematopoietic cells generated greater number of GFP(+) cardiomyocytes than Lin(-/low)CD45(-) mesenchymal cells (37.0+/-23.9 vs 0.00+/-0.00 GFP(+) cardiomyocytes per a recipient, P = 0.0095). The number of transplanted purified HSCs (Lin(-/low)Sca-1(+) or Lin(-)Sca-1(+)c-Kit(+) or CD34(-)Lin(-)Sca-1(+)c-Kit(+)) showed correlation to the number of GFP(+) cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP(+) cardiomyocytes per injected cell dose was greatest in CD34(-)Lin(-)Sca-1(+)c-Kit(+) recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP(+) cardiomyocytes than common lymphoid progenitors (12.8+/-10.7 vs 0.67+/-1.00 GFP(+) cardiomyocytes per a recipient, P = 0.0021). In CFP recipients, all GFP(+) cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients.
CONCLUSIONS/SIGNIFICANCE: Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.
明确鉴定与心肌发生相关的细胞类型和机制对于有效的心脏再生医学至关重要。我们旨在鉴定能够生成心肌细胞的细胞群体，并阐明供体标记物（+）心肌细胞的产生是否需要 BM 来源的细胞与受者心肌细胞之间的细胞融合。
方法/主要发现：从绿色荧光蛋白（GFP）小鼠中纯化 BM 干细胞/祖细胞，并将其移植到 C57BL/6 小鼠或青色荧光蛋白（CFP）转基因小鼠中。从脐血中纯化人造血干细胞（HSC），并将其移植到免疫缺陷 NOD/SCID/IL2rγ（null）小鼠中。通过免疫荧光染色后共聚焦显微镜分析心脏组织中 GFP（+）细胞的心肌细胞抗原性。通过线性解混分析区分 GFP（+）供体衍生细胞、GFP（+）CFP（+）融合细胞和 CFP（+）受者衍生细胞。通过实时定量聚合酶链反应评估异种受者心脏中人类心肌细胞基因的表达。在 C57BL/6 受者中，Lin（-/low）CD45（+）造血细胞比 Lin（-/low）CD45（-）间充质细胞产生更多的 GFP（+）心肌细胞（每个受者 37.0+/-23.9 与 0.00+/-0.00 GFP（+）心肌细胞，P=0.0095）。移植的纯化 HSC（Lin（-/low）Sca-1（+）或 Lin（-）Sca-1（+）c-Kit（+）或 CD34（-）Lin（-）Sca-1（+）c-Kit（+））数量与 GFP（+）心肌细胞数量呈正相关（每个细胞群中 P<0.05），并且在 CD34（-）Lin（-）Sca-1（+）c-Kit（+）受者中，每个注射细胞剂量的 GFP（+）心肌细胞发生率最高。在造血祖细胞中，总髓系祖细胞比共同淋巴祖细胞产生更多的 GFP（+）心肌细胞（每个受者 12.8+/-10.7 与 0.67+/-1.00 GFP（+）心肌细胞，P=0.0021）。在 CFP 受者中，检查的所有 GFP（+）心肌细胞均共表达 CFP。在一些异种受者的心脏组织中检测到人类肌钙蛋白 C 和肌球蛋白重链 6 转录本。
结论/意义：我们的结果表明，HSC 通过融合依赖的机制通过髓系中间产物产生心肌细胞。使用髓系衍生物作为供体细胞可能允许更有效的基于细胞的心脏修复治疗。
