Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) Flow cytometry analysis of composition of freshly dissociated hFKs containing NCAM1 + EPCAM - early epithelial progenitors as well as NCAM1 + EPCAM + committed early differentiated epithelium. Distribution of gestational age of hFKs received for research; weeks 17–19 were the most common. ( B ) Morphological difference between NCAM1 + PAX2 + early developing nephrons and EPCAM + mature differentiated epithelial. NCAM1 + structures are aggregates, while EPCAM + structures are more convoluted and tubular in nature. ( C ) hFKOs with EPCAM + VIM + cells undergoing mesenchymal-to-epithelial transition (MET) and also MEIS1 + fetal kidney stromal cells surrounding the epithelial structures. ( D ) Mature proximal tubule structures in hFKOs expressing HNF1B, CDH6, and LTL. ( E ) Left, human embryonic stem cell-derived kidney organoids stained for LTL and synaptopodin (SYNPO). Middle, P0 hFKOs with podocytes expressing SYNPO accompanied by LTL + proximal tubules. Left, P2 hFKOs expressing SYNPO. ( F ) ECAD + GATA3 + connecting tubules in hFKOs. ( G ) CD31 + endothelial cells form small blood vessels in EPCAM + hFKOs. ( h ) hFKOs consist of multiple lineages, including WT1 + CDH6 + proximal tubules and JAG1 + medial and distal lineages.

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Staining

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: Similarly, to the native tissue, hFKOs harbor. ( A ) Nephron progenitor cells which are NCAM1 + SIX2 + , scale bars: fetal tissue—50 μm, hFKOs—20 μm. ( B ) Early nephron epithelium which expresses NCAM1 + PAX2 + , enabling the growth and proliferation of these organoids. Scale bars: fetal tissue—20 μm, hFKOs—20 μm. ( C ) The proximal tubule has a differentiation axis which involves early WT1 expression, followed by CDH6 and ultimately LTL. These markers are present in human fetal kidney organoids, suggesting that differentiation states are conserved in vitro. scale bars: fetal tissue—20 μm, hFKOs—20 μm. ( D ) In addition to proximal tubules, MUC1 + and ECAD + distal tubule structures are present in culture. Scale bars: fetal tissue—20 μm, hFKOs—20 μm. ( E ) The C-shape is a hallmark developmental structure in nephron differentiation, in which nephron segmentation begins. Scale bar: 20 μm. ( F ) The segmentation and patterning orchestrated by the proximal WT1, Medial JAG1 and Distal LHX1 markers are recapitulated in hFKOs. Scale bar: 20 μm. .

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) heatmap comparing expression of key NPC, Renal vesicle/comma-shape body (RVCSB), proximal (SSBpr), medial-distal (SSBm_d) and podocyte (SSBpod) compartments of s-shaped bodies (SSBs) and ureteric bud/collecting duct (UBCD) markers in P0 (Freshly dissociated, embedded and cultured for 2–3 weeks) and P2 hFKOs (Organoids which were passaged twice and were cultured for 8 weeks in total). ( B ) Ontology analysis of P0 (Red) and P2 (Blue) hFKOs, processes of nephron morphogenesis and renal system development are evident in P0, while P2 is characterized by tube development and morphogenesis, in line with expected maturation and differentiation of the hFKO culture. ( C ) hFKOs cultured from week 18 of gestation for 5 months to p6 (aligning in time with the antepartum kidney, prior to birth), exhibit a maturation process commencing with a high expression of developmental genes such SIX2, PAX2, LHX1, HNF1B , and WT1 and progressing to a mature tubular kidney epithelium expressing PAX8 and CDH1 , as well as segment-specific markers such as CUBN (proximal) and MUC1, KRT8 (distal). While developmental are highly expressed in earlier passages in contrast to mature genes and vice versa, these genes are still expressed in higher passages, hence the trapezoid shape of the expression gradient across time. ( D ) Heatmap of the developmental program undergoing in hFKOs. P0 hFKOs show high expression levels of developmental genes such as JAG1, CDH6, WT1, EYA1, NCAM1, SIX2, SIX1 . Late P6 hFKOs exhibit higher levels of maturation markers such as PAX8 , CHD1, EPCAM , as well as segment-specific markers of proximal ( CUBN ) and distal ( KRT18, IRX2, MUC1 ) segments. ( E ) Ontology analysis of Early (P0) and Late (P6) hFKOs: kidney and renal system development are evident at P0, whereas P6 is characterized by epithelial cell development and renal absorption, in line with the expected maturation and differentiation of the hFKO culture. ( F ) P2 hFKOs, contain NCAM1 + PAX2 + tubular epithelium progenitors (Scale bar 20um), EPCAM + VIM + (Vimentin) tubular epithelium with ongoing differentiation and MET. CDH6 + WT1 + structures containing early proximal tubules and hFKOs with early distal, LHX1 + , and proximal, WT1 + , compartments of the SSB. Scale bars, 50 μm. ( G ) Scheme of isolation of NCAM1 + cells, by magnetic cell sorting (MACS) from P2 hFKOs, reveal the ability of these cells to form organoids ( H ) Brightfield images of NCAM1-negative fraction, devoid of newly grown hFKOs (left, scale bar 100um), Widefield NCAM1-positive fraction, giving rise to hFKOs (middle, scale bar 200 μm, n = 4), Zoomed image of a NCAM1 + derived kidney organoids (right, scale bar 100 μm). ( I ) IF of NCAM1 + -derived kidney organoids containing LTL + MUC1 + HNF1B + proximal–distal tubular epithelium (left, scale bar 50 μm), additional NCAM1 + PAX2 + epithelial progenitors (Middle, scale bar 20 μm) and EPCAM + NCAM1 + nascent nephron epithelium (right, scale bar 20 μm). ( J ) Heatmap of developmental genes, comparing expression between iPSC-KOs at early (D10) and late (D18) stages of differentiation, to hFKOs at P0 and P6. ( K ) Ontology analysis of Early hFKOs (P0, blue) vs differentiated iPSC-KOs (iPSCBD18, red): enrichment of genes correlating to urogenital and renal system development as well as epithelial cell proliferation and kidney development, while mitosis-related processes are evident in iPSC-KOs. ( L ) Ontology analysis of Late hFKOs (P5 and P6, blue) vs differentiated iPSC-KOs (iPSCBD18, Red): After 5 months of culture, P6 hFKOs are enriched with genes governing epithelial cell development as well as metanephric nephron epithelium development. Similar to the comparison to early hFKOs, iPSC-KOs are enriched with proliferation-related genes that regulate cell division and growth. .

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Isolation, FACS, Derivative Assay, Comparison

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) Immunofluorescent imaging of human embryonic stem cell-derived kidney organoids (hESC-KOs), expressing many NCAM1+ and PAX2+ structures, containing nephron progenitor cells at Day 14 of differentiation. These structures become less abundant as the culture progresses, even after an additional two days (D16). As the differentiation progresses, PAX2+ cells become organized into tubular structures and decrease in number. scale bar 50 μm. ( B ) IF imaging of hiPSC-derived kidney organoids. NPC populations co-expressing NCAM1 + PAX2+ diminish as the organoid differentiates and NCAM1 cells become unorganized and are more abundant in the stroma of the organoid. ( C ) in P2 hFKOs, after 8 weeks of culture, NCAM1 + PAX2+ co-expression is evident in epithelial progenitors. Scale bar 20 μm. ( D ) Flow cytometry of positive and negative fractions of NCAM1+ after magnetic sorting.

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Imaging, Derivative Assay, Expressing, Flow Cytometry

Journal: The EMBO Journal

Article Title: Human fetal kidney organoids model early human nephrogenesis and Notch-driven cell fate

doi: 10.1038/s44318-025-00504-2

Figure Lengend Snippet: ( A ) UMAP clustering of P0 hFKOs reveal 16 clusters: nephron progenitors (NPCs), renal vesicles (RVs), renal vesicles and comma-shaped bodies (RVs-CSBs), early podocytes (Early Pod), podocytes (Pod-1, Pod-2), proximal tubules (PT), early distal tubule/connecting tubules (Early DT/CNT), distal/connecting tubules (DT/CNT), TOP2A + KI-67 + cells (Proliferating), stressed cells (Stress), stromal cells (Stroma 1, Stroma 2, Stroma 3), collecting ducts (CD), and endothelial cells (Endo). ( B ) Violin plots of the expression of key markers used to characterize clusters. ( C ) Feature plots of markers depicting development and regeneration states, NPCs ( LYPD1 , PAX2 , NCAM1), differentiating epithelium ( PAX8 , LHX1 , JAG1 ) differentiated epithelium ( EPCAM ), and cells with regenerative capacity ( CD24 ). ( D ) Projection of hFKOs on PDX-Wilms tumors, NPCs, and proliferating cells from hFKOs match blastema tumor cells. ( E ) UMAP clustering of the nephrogenic compartment of hFKOs after sub-setting. ( F ) Pseudotime plot of nephrogenic compartment, starting at NPCs and bifurcating into podocytes and tubular lineages. ( G ) UMAP clustering of developmental markers across pseudotime axis in nephrogenic compartment of hFKOs. .

Article Snippet: hFKOs were grown for two passages (P2, 8 weeks of culture) and dissociated into single cells, which were isolated by positive selection of NCAM1 (CD56 Microbeads, 130-097-042, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 4: Porcine antigen presenting cells (pAPCs) induce proliferation of human B cells: Human peripheral blood lymphocytes were separated into CD19+ve (B-cell enriched) and CD19–ve (B-cell depleted) fractions by magnetic bead separation, then labeled with CFSE and cocultured for 96 h with no addition, 1 : 40 pGM-CSF APCs (GM-pAPCs), or 1 : 40 pGM-CSF + pIL-4 APCs (GM-IL-4-APCs). Cells were surface stained for CD19 (CD19-PE) and proliferation was measured by flow cytometry on the basis of CFSE dilution. Examples of analysis of B-cell enriched and B-cell depleted populations (following PI exclusion) are shown for no addition and pAPC coculture. B-cell enriched fractions (upper panels) were predominantly CD19+ve and demonstrated CFSE dilution only in the presence of pAPCs. B-cell depleted fractions (lower panels) were predominantly CD19–ve and demonstrated negligible cell division under all conditions. When results were expressed graphically (lower figure) as mean percent divided ± SD of triplicate samples for each condition, there were significant rates of cell division in the B-cell enriched but not the B-cell- depleted populations compared with control conditions. yp < 0.05 for values greater than the value with no addition.

Article Snippet: Purification of B cells was carried out by magnetic separation using antihuman CD19 microbeads and a magnetic separation column according to the manufacturer’s instructions (Miltenyi Biotech Inc., Auburn, CA).

Techniques: Labeling, Staining, Flow Cytometry, Control

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 5: Porcine antigen presenting cells (pAPCs) induce proliferation of murine B cells: Murine whole lymph node cells from lipopolysaccharide-responsive (C3H/HeN) and LPS-unresponsive (C3H/HeJ) strains were labeled with CFSE and cocultured for 72 h with irradiated pAPCs generated using pGM-CSF alone (pGM-CSF APCs) or pGM-CSF combined with pIL-4 (pGM-CSF/ pIL-4 APCs) at ratios of 40 : 1. Division of B-cell and T-cell subsets was detected by three-color flow cytometric analysis using PI exclusion, surface staining for CD19 (B cells) or Thy 1.2 (T cells), and CFSE fluorescence dilution. Examples of PI-gated dot-plots and CFSE fluorescence histograms gated on B cells (aCD19-PE: left upper panels) or T cells (aThy 1.2-PE: left lower panels) are shown for C3H/HeN cells cultured with No APCs, pGM-CSF APCs or pGM-CSF/pIL-4 APCs. Results are shown graphically for B cells (CD19+ve/PI–ve: upper graph) and T cells (Thy 1.2+ve/PI–ve: lower graph) and are expressed as the mean– SD percentage of viable CD19 or Thy1.2-positive cells that had undergone division on the basis of reduced CFSE fluorescence. Co-culture of lymph node cells from both murine strains was associated with significantly increased rates of B-cell division compared with control conditions (no APCs). pGM-CSF/pIL-4 APCs induced significantly greater B-cell proliferation compared with pGM-CSF APCs. Rates of B-cell division for APC-stimulated cultures were between 65% and 80%. Significant increases in T-cell division compared with control conditions also occurred for C3N/HeN and C3N/HeJ strains using both types of pAPC but were of low magnitude (between 20 and 35%). Stimulation of lymph node cells from which B cells had been depleted (T-cell enriched: inset graph) did not alter the magnitude of the T-cell response to pAPC stimulation. Basal B-cell and T-cell proliferation was greater for C3H/HeJ compared with C3H/HeN lymph node cells but results were qualitatively similar for both strains. *p< 0.05 compared with control conditions (No APCs), yp < 0.05 for pGM-CSF/pIL-4 APCs compared with pGM-CSF APCs.

Article Snippet: Purification of B cells was carried out by magnetic separation using antihuman CD19 microbeads and a magnetic separation column according to the manufacturer’s instructions (Miltenyi Biotech Inc., Auburn, CA).

Techniques: Labeling, Irradiation, Generated, Staining, Fluorescence, Cell Culture, Co-Culture Assay, Control

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 6: Porcine antigen presenting cells (pAPCs) migrate to lymph nodes and induce B-cell expansion in vivo: (A) Flow cytometric analysis of cells from left and right inguinal lymph nodes 6 h following subcutaneous inoculation of 3 105 CFSE- labeled pGM-CSF APCs to the left inguinal region and PBS to the right inguinal region. Fluorescent cells are present in the node draining the left but not the right inguinal region. (B) Groups of five B6 mice were inoculated subcutaneously with 50 mL of PBS to the right inguinal region or with one of three doses of pGM-CSF APCs (2 105, 2 104, 2 103) to the left inguinal region. Five days later all nodes were individually dissected and total cells counted. Surface staining for CD19 and flow cytometry was used to determine the B-cell percentage for each node. Results (expressed as mean ± SD, PBS vs. pAPC inoculations) for each group are shown graphically for B-cell percentage (% CD19+ve, left graph) and for total B-cell numbers per node (right graph). A significant, dose-dependent increase in B-cell percentage and total B-cell numbers occurred in lymph nodes draining pAPC inoculation sites. yp < 0.05 for pAPC vs. PBS.

Article Snippet: Purification of B cells was carried out by magnetic separation using antihuman CD19 microbeads and a magnetic separation column according to the manufacturer’s instructions (Miltenyi Biotech Inc., Auburn, CA).

Techniques: In Vivo, Labeling, Staining, Flow Cytometry