Detection of Hematopoietic Stem Cell Transcriptome in Human Fetal Kidneys and Kidney Organoids Derived From Human Induced Pluripotent Stem Cells
Authors: Frank Griscelli
Christophe Desterke
Jin Wook Hwang
Annelise Bennaceur-Griscelli
Ali G Turhan
Julien Loisel-Duwattez
Journal: Frontiers in Cell and Developmental Biology (2021)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: IPSC; fetal kidney; hematopoietic stem cell; organoid; transcriptome.
Affiliations: INSERM U935/UA09, Université Paris-Saclay, Villejuif, France., ESTeam Paris Sud, Université Paris Sud, Villejuif, France., INSERM U1195, AP-HP, Service de Neurologie, Faculté de Médecine, Hôpital Bicêtre, Université Paris-Saclay, Le Kremlin-Bicêtre, France., INGESTEM National IPSC Infrastructure, Villejuif, France., Division of Hematology, AP-HP Paris Saclay, Le Kremlin-Bicêtre, France.

Abstract:  Background: In mammalians, hematopoietic stem cells (HSCs) arise in the dorsal aorta from the hemogenic endothelium, followed by their migration to the fetal liver and to the bone marrow. In zebrafish, the kidney is the site of primary hematopoiesis. In humans, the presence of HSCs in the fetal or adult kidney has not been established. Methods: We analyzed the presence of HSC markers in the human fetal kidneys by analysis of single-cell datasets. We then analyzed in kidney organoids derived from induced pluripotent stem cells (iPSCs) the presence of hematopoietic markers using transcriptome analyses. Results: Twelve clusters were identified as stromal, endothelial, and nephron cell type-specific markers in the two fetal stage (17 weeks) kidney datasets. Among these, the expression of hematopoietic cells in cluster 9 showed an expression of primitive markers. Moreover, whole transcriptome analysis of our iPSC-derived kidney organoids revealed induction of the primitive hematopoietic transcription factor RUNX1 as found in the human fetal kidney cortex. Conclusion: These finding support the presence of cells expressing HSC transcriptome in the human kidney. The mechanisms of the appearance of the cells with the same transcriptional features during iPSC-derived kidney organoid generation require further investigation. 

Retracted: No

PubPeer comments:
 I have some serious concerns about this paper and feel that there is not enough information about the methods and organoid model shown in this paper to warrant publication. I have outlined my concerns below: (1) Firstly, the authors methods and key resource table do not match up. The generation of their kidney organoids is not convincing since they have managed to produce  kidney organoids without any directed differentiation in Essential E8 medium which does not contain any growth factors that are necessary for formation of intermediate mesoderm and technically should keep the cells within their aggregate, pluripotent. If this is a new method, I would expect extensive characterisation of the organoids by immunofluorescence of key markers (proximal tubule, distal tubule, podocytes, renal stroma) as well as more extensive ultrastructural characterisation. In addition, there is no evidence of biological replicates. (2) The nephrin staining is not convincing and the antibody (ab85379) that they used for immunofluorescence has not been validated for IF, only IHC-P (According to the manufactures guidelines). I would like to see biological replicates and antibody validation. (3) The authors identified that haematopoietic markers are upregulated in human fetal kidney. The authors failed to realise that in the single cell dataset from Lindstrom et al 2018 also contains immune cells and is not just comprised of kidney cortex cells. Looking at the original data, Lindstrom et al have identified a cluster of CXCR4+SRGN+RUNX1+SPI+ immune cells (Cluster 1, Lindstrom et al 2018). This is not new. The authors conclude that the human fetal kidney contains cells harbouring a hematopoetic transcriptome..this is obvious considering the presence of immune cells within the dataset.. ![file] ![file] ![file] ![file] (4) The authors also identified these genes upregulated in their iPSC-derived kidney organoids during the differentiation process using microarray analysis. The authors have not clearly presented this data and instead, most of the data in this paper revolves around the reanalysis of scRNAseq data from Lindstom et al. Figure 4 is not convincing and I see no evidence of these genes being upregulated. At what point during the differentiation were these genes upregulated? Where is the functional enrichment data? In addition, lack of supplemental data for the microarray experiment is also concerning. (5) Have the authors considered that master regulators of hematopoiesis are also regulators of nephrogenesis? RUNX1 promotes TGFbeta induced EMT in renal fibrosis and is also expressed in kidney tubules. CD34 for example is also involved in epithelial lumen formation during nephrogenesis. Overall I think that the suggestion of a kidney microenvironmental HSC niche is weak. , 
 (3) Single cell cluster 1 in (Cluster 1, Lindstrom et al 2018: PMID: 29870722) is not immune cells but NPC prolif. in the original paper from Lindstrom et al 2018 there is no presentation of the immune cell cluster. This peer review did not provide figure from original paper from Lindstrom et al 2018 but a new analyse which reproduce what we seen in the paper that in presented (PMID: 34178994).Based on which immune markers the immune cell cluster was done in this peer review? On the figure provided in this peer revew, as you seen CXCR4 is expressed in other cell lineage that cell was named immune cells by the provider!?! (4) the transcriptome analysis of the organoid was provided in Figure 4 of the paper (PMID: 34178994) (5) in CD45+ cluster (c9) of single cell analysis provided on analysis from paper PMID: 3417899, expression of SPI1 transcription factor which are considerate as a master regulator of human adult hematopoiesis also CXCR4 receptor is known to be necessary for migration of HSC from fetal liver to bone marrow. Concerning RUNX1 that was found expressed in the kidney IPSC derived organoid, it is another master regulator of hematopoieisis well know to act in acute myeloid leukemia. ,