In recent years stem and progenitor cells have emerged as a promising regenerative medicine tool. Their characteristics of self-renewal and pluripotency suggest that stem cells may be useful to repair injured tissue and reconstruct damaged organs.5
AF recently raised the interest of scientists as an alternative source of pluripotent cells. Due to its contact with the developing fetus AF contains abundant suspended cells, which have long been used for diagnostic purposes. The presence of mature cells in AF, such as smooth muscle cells, osteoblasts and lung epithelial cells, has long been known.6
More recently previously described stem cells were identified.2
These AFSs are easily obtained, largely avoid the ethical concerns associated with embryonic stem cell use and propagate in culture without difficulty, maintaining their pluripotential capacity. However, they comprise no more than 1% of the total AF cellular population.
Our main objective was to better characterize the remaining cells in AF, focusing on cells belonging to the renal lineage. We identified cells expressing markers of all 3 germ layers, mesenchymal and hematopoietic, progenitors of different organs and particularly cells with specific renal characteristics, including podocyte precursors, epithelial tubular cells and mesangial cells.
Human embryo development follows a precise timetable during gestation. We hypothesized that cells in the AF are committed to various organs at different gestational time points. In the time range investigated younger samples expressed more frequently and at higher levels markers related to mesodermal and endodermal germ layers while in older samples these markers decreased in frequency and quantity due to AF cell differentiation with time and the different degrees of maturation of cells detaching from the fetus. The expression of organ specific markers also increased with gestational age, presumably due to organogenesis maturation. Since most AF volume derives from fetal urine,1
it is reasonable to assume that kidney progenitor cells are a major constituent of AF. The expression of renal markers such as PAX-2, LIM-1
, nephrin, PDGFRA, TRKA
, E-cadherin, CD24
and OB-cadherin showed a clear increase by the end of week 17 of gestation ().
After noting that kidney progenitors were present in the total AF cell population we selected a specific AF population based on in vivo studies7
showing CD24 and OB-cadherin co-expression in the developing kidney, particularly in MM, which together with the ureteral bud gives rise to the mature kidney. We called this population MM-like cells (CD24+OB-cadherin +) (). When MM is induced by the ureteral bud, the expression of GDNF, LIM-1, PAX-2, BMP-2
and other genes becomes evident, as also expressed in our CD24+OB-cadherin+ population. Subsequently with kidney maturation gene expression varies and begins to be restricted to specific cell lineages. Each cell lineage acquires characteristic traits that are driven by specific gene expression and indicated by surface markers such as E-cadherin (mesenchymal-to-epithelial transition cells),8
podocalyxin (mature podocytes),9
TRKA (stromogenic cortical mesenchymal cells)10
and PDGFRA (mesangial cells).10
Thus, these surface markers were used to perform additional immunoselection from the initially isolated CD24+OB-cadherin+ population.
Genes and proteins such as GDNF, WT-1, LIM-1 and PAX-2 in the 5 CD24+OB-cadherin+ subpopulations determine the fate of renal cell types. We also investigated OCT-4 expression to determine whether subselected cells still had multidifferentiation potential. During kidney development organ specific precursors go through different stages of differentiation to attain the mature state. During this maturation process pluripotent genes are not turned off suddenly and all maturity specific genes are not suddenly turned on. In the intermediate state co-expression of the 2 gene types occurs. Real-time PCR confirmed a specific temporal pattern of renal marker expression in each of the 5 subpopulations derived from CD24+OB-cadherin + cells ().
Genes expressed in AKPC subpopulations on real-time PCR
An interesting result was revealed by WT-1 expression, which was expressed in vivo in the metanephros in the proximal part of the S-shaped body and then exclusively in mature podocytes. Of all isolated subpopulations only nephrin+ and podocalyxin+ cells expressed WT-1
, indicating that they are indeed podocyte precursor cells.11
Genes such as PAX-2
were not expressed in podocalyxin+ selected cells but were evident in nephrin+ cells, suggesting that nephrin+ cells may represent a more immature podocyte lineage than podocalyxin expressing cells. This concept was also supported by the finding that podocalyxin+ cells did not express OCT-4
but nephrin+ cells expressed OCT-4
at high levels. Cultured podocalyxin+ cells showed the typical morphology of cultured human podocytes with primary processes similar to those described by Vogelmann et al ().12
In the other populations WT-1
was not expressed, showing no commitment to podocyte differentiation. The expression pattern of PDG-FRA+ cells with PAX-2
expression and absent LIM-1
suggested commitment to the nephrogenic lineage but not current mesenchymal-to-epithelial transition cells. OCT-4
was highly expressed, a characteristic shared with the TRKA+ population. The TRKA+ population was highly positive for PAX-2, LIM-1
and TRKA. TRKA
, which is expressed only in developing kidney stromogenic cortical mesenchymal cells,9
suggests TRKA+ cell commitment. E-cadherin+ cells did not express PAX-2
but rather showed LIM-1
and E-cadherin expression, suggesting incomplete commitment to the nephrogenic differentiation pattern.12
Morphology of AKPC podocalyxin+ cells cultured with Chang's medium shows primary processes elongating from cell body. Reduced from ×10.