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Abstract: TH-PO643

Identification of ROBO2 and Integrin B4 as Potential Surface Markers for the Isolation of Live Nephrogenic Cells from Human Fetal Kidneys

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

  • Petrosyan, Astgik, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Marcheque, Julia, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Thornton, Matthew Edward, University of Southern California, Los Angeles, California, United States
  • Grubbs, Brendan, University of Southern California, Los Angeles, California, United States
  • Perin, Laura, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Da Sacco, Stefano, Children's Hospital Los Angeles, Los Angeles, California, United States
Background

In the developing kidney, the formation of new nephrons relies on a small population of self-renewing nephrogenic progenitors (NP) characterized by co-expression of SIX2 and CITED1. Despite their essential role in renal formation and maturation, identification of surface markers that can facilitate their isolation has not yet been successful. We report here the isolation of a live cell population with NP traits from human fetal kidneys (hFK) using a novel combination of surface markers.

Methods

We have previously reported the isolation of NP cells from hFK cells using RNA probes. Based on our RNA-seq performed on these cells we have identified ROBO2 and Integrin B4 as potential surface markers for direct isolation of NP of human origin. ROBO2+IntegrinB4+ cells were isolated using FACS and RNAseq analysis was immediately performed to characterize their gene expression and evaluate their genetic profile. Data were compared with the SIX2+CITED1+ cells isolated by RNA-probe. Dissociation/reaggregation assays were performed to confirm their nephrogenic traits. Potential for long term expansion was also explored.

Results

Expression of ROBO2 and IntegrinB4 was histologically confirmed within the cap mesenchyme during development in 17-week hFK . FACS of ROBO2+IntegrinB4+ cells from hFK confirmed enriched expression of SIX2 and CITED1 in more than 90% of the cells. RNASeq analysis confirmed expression of genes including SIX2, CITED1, SIX1, PHF19, OSR1 and EYA1 suggesting a nephrogenic signature. Expression of human NP markers was validated by flow cytometry. NP showed the ability to integrate in developing renal structures expressing nephrogenic markers when co-cultured with dissociated/re-aggregated hFK. Optimal culture conditions for long-term expansion without loss of NP traits were also successfully established.

Conclusion

Our preliminary results suggest that ROBO2 and IntegrinB4 are potential candidates as surface markers for the direct isolation of cells with nephrogenic characteristics, including the expression of SIX2 and CITED1, from hFK without the use of any genetic manipulation. This system represents a novel method of isolating a pool of NP that can be applied towards studies of renal cell specification, thus increasing our knowledge of human renal development

Funding

  • Private Foundation Support