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

Filipa M. Lopes, Ioannis Bantounas, Alexandra Sarov, Adrian S. Woolf, Susan J. Kimber

Posted on: 16 January 2026

Preprint posted on 16 January 2026

Immune cells for nephron development; tweaking the organoid recipe! In the preprint highlighted here, the authors use human PSC-derived organoids to evaluate the contribution of macrophages to kidney development.

Selected by Theodora Stougiannou

Categories: bioengineering, cell biology

Figure 1 ‘[Human pluripotent stem cell-derived macrophages modify development of human kidney organoids]’ Study Summary/ Created in BioRender. Stougiannou, T. (2025) https://BioRender.com/ux5t0tf, Human pluripotent stem cell-derived macrophages modify development of human kidney organoids, Lopes FM, Bantounas I, Sarov A, Woolf AS, Kimber SJ, 2025, Illustrations created in BioRender for the purposes of this preprint highlight only [Organoid Figure 3: Created in BioRender. Stougiannou, T. (2025) https://BioRender.com/3ycd0q8, Organoid Figure 4: Created in BioRender. Stougiannou, T. (2025) https://BioRender.com/Az6cbwl]

Background: Kidney development involves several stages; during the last stage, also known as the metanephros stage, the so-called ureteric bud (UB) derived from structures present in previous developmental stages branches into the nearby, developing metanephric mesenchyme (MM). It is during this stage that nephrogenesis occurs, with two main types of epithelia derived from two distinct sources; nephron epithelia derived from the MM that will eventually give rise to glomerular podocytes, the Bowman capsule as well as the proximal and distal tubules, and collecting ducts derived from the UB. In turn, all these populations are derived from intermediate mesoderm [1]. The kidney also contains immune cell populations, such as local macrophages, which usually develop from hematopoietic precursors present in the embryonic yolk sac and liver. These macrophages have been shown to contribute to metanephric kidney development, as they contribute to clearance of apoptotic cells and vascular development, at least in animal models. The contribution of macrophages to kidney development in humans, however, remains unclear [2].

The study by Filipa M. Lopes et al. [2] seeks to use pluripotent stem cell (PSC)-derived nephron organoids to evaluate the contribution of PSC-derived macrophages to nephron development. Macrophages at early/later maturation stages, at concentrations of either 1%, 5% or 20%, were generated and combined with metanephric precursors to evaluate their effect on metanephric organoid development.

Key aspects of the study:

Human PSCs were initially transduced with lentiviral vectors carrying the green fluorescent protein (GFP) gene construct and were then subjected to a differentiation protocol included a hematopoietic stem cell (HSC) intermediate, eventually leading to macrophage derivation:
1. Mature macrophages stain positive for markers such as 25f9, CD74 and CD81 (markers found in resident kidney macrophages), CD68 (marker found in human fetal kidneys).
Human PSCs were subjected to a differentiation protocol aimed at deriving nephron organoids; once nephron precursor cells were generated during the course of the protocol, they were mixed with PSC-derived macrophages:
1. As nephrogenesis proceeded within organoids, visual identification of macrophages became difficult; immunostaining against CD68 helped identify macrophages in the organoids as development progressed.
2. In these composite organoids, macrophages were identified in between the tubules and around the glomeruli.
3. Organoid areas occupied by vascular structures measured to about 1% of total area, with this value not significantly changing in organoids generated with 1%, 5% or 20% early-stage macrophages; effects of late-stage macrophages depended on concentration, with 1% late-stage macrophages reducing total area of vascular structures compared to 5% late-stage macrophages.
4. There were rare occurrences of glomerular tufts incorporating CD31/PECAM-1+ endothelial cells assembling into capillary-like structures; although no significant change in the proportion of such glomeruli were identified in organoids with added macrophages, the overall mass of glomeruli as denoted by synaptopodin+ staining, seemed to increase by about 3-fold with 5% or 20% early-stage macrophages. Conversely, with addition of 1% late-stage macrophages, overall mass of glomeruli seemed to decrease.

Use of 20% early-stage or late-stage macrophages led to smaller final organoid size; in addition, with 20% late-stage macrophages disruption in the medulla formations occurred, with emergence of atypical tissues composed of stromal-like and immature blastema-like cells.

Why this work is interesting: While the contribution of macrophages to the development of the kidney has been evaluated in animal models, this has not been sufficiently explored within the context of human development, possibly owing to the ethical considerations associated with human fetal tissue experimentation. This study uses human PSC-derived organoids to explore this question, essentially pointing to the importance of macrophages, when provided at the appropriate population levels, to contribute to physiological nephron tissue development.

References:

[1] Seely JC. A brief review of kidney development, maturation, developmental abnormalities, and drug toxicity: juvenile animal relevancy. J Toxicol Pathol 2017;30:125–33. https://doi.org/10.1293/tox.2017-0006.

[2] Lopes FM, Bantounas I, Sarov A, Woolf AS, Kimber SJ. Human pluripotent stem cell-derived macrophages modify development of human kidney organoids 2025:2025.11.07.687192. https://doi.org/10.1101/2025.11.07.687192.

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Author's response

Susan J. Kimber, Filipa M. Lopes shared

What are some attributes of macrophages that make them well-suited to aid the development of the embryonic metanephros?

Developing human kidneys contain macrophages, but their possible roles there are unclear, in large part because of the challenges of undertaking functional studies on human fetal tissues.

Murine studies show that developing murine metanephric kidneys contain macrophages. They enter the murine kidney via the yolk sac and after E13,5 from the fetal liver, from haematopoietic stem cells and increasingly in later developmental, from circulating monocytes. In animal experimental models, various roles have been attributed to macrophages including clearance of apoptotic cells, enhancing vessel morphogenesis and stimulate branching morphogenesis. Macrophages have also been suggested to be involved in lymphatic development. There is good evidence that monocyte-derived macrophages can take on tissue-specific residency functions given the right environmental cues in the developing kidney, where monocyte derived macrophages become tissue resident kidney macrophages.

Human kidney organoids can be really useful for understanding normal kidney development and congenital kidney disease. Single cell analysis in multiple labs does not indicate tissue resident macrophages are present in pluripotent stem cell derived kidney organoids. Importantly, both vascular development and ureteric bud derivative branching are immature or lacking in human hPSC-kidney organoids.

We need human kidney organoids to be as authentic as possible if the model is to tell us what goes wrong in early kidney development in the presence of a mutation.

Do you think there might be other immune cell types to explore that may have similar contributions?

That’s an interesting question; probably other immune cohorts do not make the same contribution. Most are involved in kidney injury or disease; for instance, neutrophils in inflammation and promoting fibrosis after injury. Lymphocytes entering through the developing lymphatic system are involved in kidney homeostasis. These cells are also likely to be important later, when lymphocytes can respond to damage or infection. In later development, innate lymphocyte populations are present and may play a developmental role. However, macrophages seem to be the most important immune cell type during differentiation and morphogenesis.

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