Fig. 1

Enhancing NK cell differentiation from hematopoietic stem and progenitor cells through osmotic pressure modulation. A A schematic illustration of the HSC-NK cell differentiation protocol beginning with CB HSCs. Following a four-day HSC expansion, HSCs were co-cultivated with OP9-DLL1-DLL4 feeder cells with optional osmotic pressure regulation via the addition of a 9% NaCl water solution (w/v) to the medium. B The expression levels of CD34, CD56, and CD3 cells both before and after a 14-day period of HSC expansion and differentiation. C Frequency of CD56⁺ CD3⁻ NK cells after 14 days of HSC expansion and differentiation (mean ± SEM, n = 4 independent experimental replicates). High osmotic pressure was manipulated by adding 22.4 μl of a 9% NaCl solution to 1 ml of medium, resulting in a final osmotic pressure of 330 mM. D Frequency of CD56⁺ CD3⁻ NK cells after 14 days of HSC expansion and differentiation under the specified osmotic pressure regulation (mean ± SEM, n = 4 independent experimental replicates). E The number of CD56⁺ CD3⁻ NK cells generated from a single HSC, as demonstrated in (D) (mean ± SEM, n = 4 independent experimental replicates). F Frequency of CD56⁺ CD3⁻ NK cells following a 14-day period of HSC expansion and differentiation. The experiments were conducted utilizing HSPCs from two distinct donors, with each experiment replicated technically twice. To achieve a final osmotic pressure of 330 mM, we introduced 22.4 μl of 1.5 M KCl, 44.8 μl of 1.5 M glucose, or 44.8 μl of 1.5 M sucrose into 1 ml of medium. G The quantity of CD56⁺ CD3⁻ NK cells generated from a single HSC, as indicated in (F), with corresponding fold changes noted. The P-values obtained from the paired Student's t-test are indicated in panels (C), (D), and (E), while panels (F) and (G) underwent a two-way ANOVA analysis