Quantification of cellular proliferation in humans is important for understanding biology and responses to injury and disease. However, existing methods require administration of tracers that cannot be ethically administered in humans. We present a protocol for the direct quantification of cellular proliferation in human hearts. The protocol involves administration of non-radioactive, non-toxic stable isotope ¹⁵Nitrogen-enriched thymidine (¹⁵N-thymidine), which is incorporated into DNA during S-phase, in infants with tetralogy of Fallot, a common form of congenital heart disease. Infants with tetralogy of Fallot undergo surgical repair, which requires the removal of pieces of myocardium that would otherwise be discarded. This protocol allows for the quantification of cardiomyocyte proliferation in this discarded tissue. We quantitatively analyzed the incorporation of ¹⁵N-thymidine with multi-isotope imaging spectrometry (MIMS) at a sub-nuclear resolution, which we combined with correlative confocal microscopy to quantify formation of binucleated cardiomyocytes and cardiomyocytes with polyploid nuclei. The entire protocol spans 3–8 months, which is dependent on the timing of surgical repair, and 3–4.5 researcher days. This protocol could be adapted to study cellular proliferation in a variety of human tissues.

人类细胞增殖的量化对于理解生物学以及对损伤和疾病的反应非常重要。然而，现有的方法需要施用示踪剂，而这在人类身上施用是不符合伦理的。我们提出了一种直接定量人类心脏细胞增殖的方案。该方案涉及对患有法洛四联症（先天性心脏病的一种常见形式）的婴儿施用非放射性、无毒的稳定同位素¹⁵富氮胸苷（ ¹⁵ N-胸苷），该同位素在 S 期掺入 DNA 。患有法洛四联症的婴儿需要接受手术修复，这需要切除原本会被丢弃的心肌碎片。该协议允许量化该废弃组织中的心肌细胞增殖。我们利用多同位素成像光谱法 (MIMS) 在亚核分辨率下定量分析了¹⁵ N-胸苷的掺入情况，并将其与相关共聚焦显微镜相结合，以量化双核心肌细胞和具有多倍体细胞核的心肌细胞的形成。整个方案持续 3-8 个月（取决于手术修复的时间）和 3-4.5 个研究日。该协议可适用于研究各种人体组织中的细胞增殖。