Isolation of clonogenic, long-term self renewing embryonic renal stem cells

Abstract

A tissue stem cell should exhibit long-term self-renewal, clonogenicity and a capacity to differentiate into the tissue of origin. Such a postnatal renal stem cell has not been formally identified. The metanephric mesenchyme (MM) of the developing kidney gives rise to both the renal interstitium and the nephrons and is regarded as the progenitor population of the developing kidney. However, isolated MM does not self renew and requires immortalization for survival in culture. Here we report the isolation and sustained culture of long-term repopulating, clonal progenitors from the embryonic kidney as free floating nephrospheres. Such cells displayed clonal self renewal for in excess of twenty passages when cultured with bFGF and thrombin, showed broad mesodermal multipotentiality, but retained expression of key renal transcription factors (Wt1, Sall1, Eya1, Six1, Six2, Osr1 and Hoxa11). While these cells did display limited capacity to contribute to developing embryonic kidney explants, nephrospheres did not display in vitro renal epithelial capacity. Nephrospheres could be cultured from both Sall1(+) and Sall1(-) fractions of embryonic kidney, suggesting that they were derived from the MM as a whole and not specifically the MM-derived cap mesenchyme committed to nephron formation. This embryonic renal stem cell population was not able to be isolated from postnatal kidney confirming that while the embryonic MM represents a mulitpotent stem cell population, this does not persist after birth.

Rapid and Efficient Differentiation of Human Pluripotent Stem Cells into Intermediate Mesoderm That Forms Tubules Expressing Kidney Proximal Tubular Markers

“…43,66 Briefly, embryonic kidneys at stage E12.5 (day of plug=E0.5) were isolated from timed pregnant females (Charles River).…”

Renal organogenesis

“…Spheres were also able to be cultured indefinitely when supplemented with conditioned media from other sphere cultures. 31 Spheres also showed multilineage potential (adipocyte, osteocyte, chrondrocyte, myotube) in vitro and retained the expression of key cap mesenchyme genes (Wt1, Sall1, Eya1, Six1, Six2, Osr1, Hoxa11, Cdh11, Gdnf ) throughout culture. In contrast, such cultures did not express renal epithelial markers or markers of renal interstitium.…”

“…3B). 31 While this remained a rare event, this is likely to reflect the nature of sphere cultures in that only a subfraction of any sphere represents the initiating stem cell. However, when these cells were delivered into models of adult acute renal injury (ischemia-reperfusion), cells were seen in the interstitial compartment and not seen to integrate into the renal epithelium (Fig.…”

“…3A). 31 Such spheres could be dissociated to single cells, with individual cells able to form secondary and tertiary spheres, validating a clonogenic capacity. Spheres were also able to be cultured indefinitely when supplemented with conditioned media from other sphere cultures.…”

Induced Pluripotent Stem Cells from Human Kidney

“…16 Although both embryonic and adult stem cells have the potential to differentiate into every cell type of the body, in the vast majority of studies, the efficiency is low and differentiation is sporadic. Therefore, with notable exceptions, 17 the road to clinical application of native stem cell-based therapy for kidney has been uncertain.…”