bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.11.07.687192

Human pluripotent stem cell-derived macrophages modify development of human kidney organoids

Lopes, F. M.·Bantounas, I.·Sarov, A.·Woolf, A. S.·Kimber, S. J.
View original source ↗Export metadata ↓

Abstract

IntroductionThe human fetal kidney contains macrophages, innate immune cells postulated to enhance its development. Macrophages have also been implicated in the pathobiology of human kidney malformations and Wilms tumour. Human pluripotent stem cell (hPSC)-derived kidney organoids contain nephrons differentiated from intermediate mesoderm-like precursors. Endothelia are present between organoid tubules but fail to efficiently populate glomeruli. These organoids lack macrophages, as expected because in vivo kidney macrophages invade from yolk sac and liver. MethodsWe hypothesised that combining hPSC-derived macrophages with hPSC-derived kidney precursors modifies nephrogenesis. Macrophages harvested at early or later maturation stages were added to kidney precursors in numbers of 1%, 5% or 20% compared with constant numbers of nephrogenic cells. ResultsNo macrophages, as assessed by CD68 immunostaining, were detected in organoids without added macrophages. In contrast, composite organoids contained macrophages located between tubules, mimicking native human fetal kidneys. Quantification of tissue macrophages at the end of 18-day organoid culture positively correlated with the numbers of macrophages added. Assessed by CD31/PECAM-1 and CD68 co-immunostaining, some macrophages were near vessels but added macrophages neither increased the proportion of vessels nor endothelial invasion of glomeruli. Early-stage macrophages significantly increased the percentage area occupied by glomeruli, as assessed by synaptopodin immunostaining. The highest macrophage numbers inhibited overall growth, assessed by organoid area, additionally generating dysmorphic tissue when later stage macrophages were added. ConclusionDepending on their maturation stage and quantity, macrophages have beneficial or harmful effects on organoids. This supports the proposition that macrophages play roles in normal and abnormal development of human kidneys.

Explore related subjects

developmental biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Lopes, F. M., Bantounas, I., Sarov, A., Woolf, A. S., Kimber, S. J.. 2025-11-08. Human pluripotent stem cell-derived macrophages modify development of human kidney organoids. https://doi.org/10.1101/2025.11.07.687192

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Extensive loss of HoxA/D genes does not disrupt anterior vertebral patterning in zebrafish

Hox genes play central roles in specifying positional identities along the vertebrate anterior-posterior axis. In mice, genetic analyses have demonstrated that Hox genes distributed among the four Hox clusters contribute to vertebral patterning, with extensive functional redundancy among paralogous genes. Our previous genetic analysis in zebrafish identified important roles for HoxB- and HoxC-related genes in specifying anterior vertebral identities, whereas the contributions of HoxA- and HoxD-related genes remained unresolved. Here, we examined adult zebrafish carrying extensive combinations of hoxaa, hoxab, and hoxda cluster deletions and generated five-gene homozygous mutants carrying frameshift mutations in hoxa3a, hoxa4a, hoxa5a, hoxd3a, and hoxd4a. X-ray micro-CT analysis revealed no obvious alterations in anterior vertebral morphology in either the compound cluster mutants or the five-gene mutants. These results indicate that HoxA/D genes make only a limited detectable contribution to anterior vertebral patterning in zebrafish. Together with our previous findings, they suggest that vertebral patterning functions are distributed unevenly among zebrafish Hox clusters, with a predominant contribution from HoxB/C-related genes.

developmental biology↗
bioRxiv2026

Developmental remodeling of ping-pong piRNA amplification in the vertebrate female germline

The piRNA pathway silences transposable elements (TEs) in the germline, and the ping-pong amplification cycle is the hallmark of this defense.In the male germline, ping-pong is most active during a meiotic window of spermatogenesis, yet its developmental profile in the vertebrate female germline remains less well explored. Most profiling has used adult ovary and mature oocytes, stages at which piRNA pathway components are reported to be low. To address this, we generated matched strand-specific RNA-seq and small RNA-seq from pre-meiotic (E10.5) and meiotic entry (E16.5) chicken ovary, used published single-cell data to track germ-cell composition across the same window, and extended the analysis to the mature chicken ovary and to zebrafish across developmental stages. Ping-pong amplification increases at meiotic entry compared to the pre-meiotic stage across TE classes. In the mature ovary, the signature weakens, and the remaining ping-pong pairs are preferentially associated with LTR/ERV retroelements. We show that activation of a meiotic entry transcriptional program in an in vitro chicken primordial germ cell model increases the fraction of piRNA-sized reads with a partner exhibiting a 10-nt 5' overlap and increases the 1U signature of piRNA-sized reads, consistent with meiotic priming promoting piRNA biogenesis. The zebrafish ovary shows a similar meiosis-associated amplification and preferential targeting of LTR/ERV retroelements at maturity, while carrying roughly 5.7-fold more TE sequences. Similar patterns in two lineages that diverged approximately 430 million years ago suggest that germline development shapes both the timing of ping-pong amplification and the TE classes preferentially associated with it.

developmental biology↗
bioRxiv2026

Allele-Resolved Hybrid Embryos Reveal the Fates of Regulatory Divergence

Developmental programs can remain conserved despite extensive regulatory divergence, but how evolved regulatory differences are transmitted through embryonic lineages remains unclear. Here we generate a time resolved, allele resolved single-cell atlas of hybrid embryogenesis between Ciona intestinalis and Ciona savignyi, enabling regulatory differences accumulated between species to be followed across defined developmental lineages. We find that allelic differences are maintained or remodeled in lineage specific ways, with their outcomes associated with regulatory origin and allele specific chromatin accessibility. Across multiple tissues, allelic divergence increases along gene regulatory network (GRN) hierarchy from upstream regulators toward downstream regulators and effector genes. In the cardiopharyngeal lineage, Foxf illustrates how allelic dominance provides partial compensation for highly divergent regulatory sequences and thereby contributes to developmental system drift. Together, our results reveal that regulatory divergence is dynamically sorted during development according to lineage context and GRN hierarchy. This lineage resolved framework provides a developmental basis for understanding how extensive regulatory evolution can accumulate while conserved embryonic programs are maintained.

developmental biology↗