#2200 Kidney stem/progenitor cells can be isolated from the urine of neonates independently of their gestational age at birth
Authors: Akalay Sara , Rayyan Maissa , Hermans Isabelle , van den Heuvel Lambertus (Bert) , Devos Timothy , Arcolino Fanny Oliveira , Levtchenko Elena 
Published: May 2024

Abstract

Background and Aims
In the human kidney, nephron structures derive from a population of SIX2 positive kidney stem/progenitor cells. These cells are only present during kidney development, which is reported to terminate at approximately 36 weeks of gestation. We have previously described a non-invasive strategy to isolate the native SIX2 positive kidney stem/progenitor cells from the urine of neonates born before 36 weeks of gestation, named the neonatal kidney/stem progenitor cells (nKSPC) [1]. In preterm neonates, nephrogenesis is still ongoing at the time of birth and continues postnatally, enabling isolation of kidney stem/progenitor cells from the voided urine. In this study, we aimed to determine the efficiency of nKSPC isolation from the urine of neonates and which gestational age (GA) results in the highest yield of nKSPC. We hypothesized that the lower the gestational age, the higher the probability of isolating nKSPC.

Method
37 fresh urine samples were obtained from 36 neonates (9 female, 27 male) at day 1 after birth at the Neonatology department of University Hospitals Leuven. Five urine samples were collected from extreme preterm (&lt; 28 weeks GA), 6 samples from very preterm (28–32 weeks GA), 18 samples from moderate to late preterm (32-37 weeks GA) and 8 samples from term neonates (&gt;37 weeks). When a sample yielded cell growth, cell colonies were subcultured to achieve clonal expansion. Cell lines were characterized for the SIX2 stem cell marker using RT-qPCR and immunofluorescence (IF) staining. SIX2 positive cell lines were further evaluated for their potential to differentiate into kidney epithelial cells (i.e. proximal tubular epithelial cells (PTEC) and podocytes) in 2D cultures using our previously established protocols [1].

Results
From the 37 urine samples collected, 28 samples yielded cell growth (76%). After subcloning, 147 cell lines were characterized for the expression of SIX2, of which 42 were SIX2 positive. Four SIX2 positive cell lines were derived from extreme preterm, 12 from very preterm, 13 from moderate to late preterm and 13 from term neonates. SIX2 positive cell lines isolated from term neonates exhibited similar cells growth and differentiation potential compared to those isolated from extreme preterm neonates. Additionally, we observed a dose-response effect with regard to expression levels of SIX2 and the differentiation potential: cell lines with higher levels of SIX2 maintained their undifferentiated, uninduced state while lower levels of SIX2 enabled successful differentiation to PTEC and/or podocytes.

Conclusion
This study demonstrates that SIX2 positive nKSPC can be isolated from the urine of neonates, independently of their GA at birth. This could indicate that nephrogenesis persists longer than what has previously been reported (i.e. 36 weeks). Furthermore, the nKSPC exhibit a dose-response effect with regard to levels of SIX2 and induction of differentiation. This effect has previously been observed in an in vivo mice model [2]. To further investigate the timing of nephrogenesis cessation, we aim to perform a SIX2 staining on human fetal kidney tissue across different GA. Furthermore, we aim to further characterize in detail the isolated nKSPC using single cell RNA sequencing.