In recent years, numerous cancers have been described as having a "cancer stem cell" (CSC) population also known as "cancer initiating cells". CSCs refer to a subset of tumour cells that has the ability to self-renew and generate the diverse cells that comprise the tumor. Their name derives from their "stem-like" properties and ability to continually sustain tumorigenesis. CSCs have the same properties that define a normal tissue adult stem cell, even if they are aberrant: self-renewal and differentiation. Renal cell carcinoma (RCC) accounts for about 3% of adult cancers and is among the 10 most common malignancies in Europe. RCC has several morphological subtypes and clear cell RCC accounts for ~80% of cases. RCC has a late diagnosis and is therapy resistant. In renal pathologies there are situation in which the presence and function of adult renal stem cells may have clinical relevance. In the last years the existence of different sources of renal stem cells has been proposed even if the phenotype of a resident stem cell of the kidney has not been exhaustively described. Even in RCC the definition of a kidney cancer stem cell may have a role for better understanding renal cell carcinoma biology. A number of approaches based on the exploitation of renal stem cell markers have allowed the prospective isolation of human renal stem cells, even if the relative promiscuity of these markers limits their usefulness when highly purified stem cells are needed. So we decided to use a functional approach for the isolation of normal and cancer stem cells of the kidney by culturing the cells in suspension, non-adherent conditions, at low density with specific growth factors. In these conditions only a little percentage of cells survives growing as spherical aggregates in suspension. We called them "nephrospheres". Using fluorescent lipophilic dyes PKH, we demonstrated the clonal origin of the spheres and the presence of a heterogeneous population inside the spheres. In fact the dye dilutes in active replicating cells while is retained in quiescent cells; we can observe in normal and cancer nephrospheres some most fluorescent cells, the quiescent stem cells, and some less fluorescent or not fluorescent cells, that are the active replicating progenitors. We performed a characterization of the nephrospheres by immunofluorescence after cytospin or FACS evaluating the expression of some epithelial and stem cell markers. By Real Time PCR we found that some genes related with stemness or involved in the maintenance of pluripotency are overexpressed in normal and cancer nephrospheres if compared with the corresponding differentiated primary cell cultures. We then evaluated the differentiative abilities of the cells derived from normal nephrospheres by culturing the cells in specific media and semisolid substrates; the cells are able to differentiate into epithelial and neuronal-like phenotype and to form tubular/glomerular-like tridimensional structures. We the isolated the stem cell population form normal nephrospheres on the basis of the PKH fluorescence. We identified 3 PKH populations: PKHhigh population, with a high level of fluorescence, PKHlow population, with a low level of fluorescence, and PKHneg population, negative for PKH. The populations were separated with cell sorting and cultivated to form spheres. Only PKHhigh cells were able to generate new spheres, demostrating that the PKHhigh cells represent the stem cell population inside the nephrospheres. Normal and cancer PKHhigh cells will be deeply characterized in the future.

(2010). Isolamento e caratterizzazione di cellule staminali adulte da rene normale e carcinoma renale. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).

Isolamento e caratterizzazione di cellule staminali adulte da rene normale e carcinoma renale

BOMBELLI, SILVIA
2010-01-21

Abstract

In recent years, numerous cancers have been described as having a "cancer stem cell" (CSC) population also known as "cancer initiating cells". CSCs refer to a subset of tumour cells that has the ability to self-renew and generate the diverse cells that comprise the tumor. Their name derives from their "stem-like" properties and ability to continually sustain tumorigenesis. CSCs have the same properties that define a normal tissue adult stem cell, even if they are aberrant: self-renewal and differentiation. Renal cell carcinoma (RCC) accounts for about 3% of adult cancers and is among the 10 most common malignancies in Europe. RCC has several morphological subtypes and clear cell RCC accounts for ~80% of cases. RCC has a late diagnosis and is therapy resistant. In renal pathologies there are situation in which the presence and function of adult renal stem cells may have clinical relevance. In the last years the existence of different sources of renal stem cells has been proposed even if the phenotype of a resident stem cell of the kidney has not been exhaustively described. Even in RCC the definition of a kidney cancer stem cell may have a role for better understanding renal cell carcinoma biology. A number of approaches based on the exploitation of renal stem cell markers have allowed the prospective isolation of human renal stem cells, even if the relative promiscuity of these markers limits their usefulness when highly purified stem cells are needed. So we decided to use a functional approach for the isolation of normal and cancer stem cells of the kidney by culturing the cells in suspension, non-adherent conditions, at low density with specific growth factors. In these conditions only a little percentage of cells survives growing as spherical aggregates in suspension. We called them "nephrospheres". Using fluorescent lipophilic dyes PKH, we demonstrated the clonal origin of the spheres and the presence of a heterogeneous population inside the spheres. In fact the dye dilutes in active replicating cells while is retained in quiescent cells; we can observe in normal and cancer nephrospheres some most fluorescent cells, the quiescent stem cells, and some less fluorescent or not fluorescent cells, that are the active replicating progenitors. We performed a characterization of the nephrospheres by immunofluorescence after cytospin or FACS evaluating the expression of some epithelial and stem cell markers. By Real Time PCR we found that some genes related with stemness or involved in the maintenance of pluripotency are overexpressed in normal and cancer nephrospheres if compared with the corresponding differentiated primary cell cultures. We then evaluated the differentiative abilities of the cells derived from normal nephrospheres by culturing the cells in specific media and semisolid substrates; the cells are able to differentiate into epithelial and neuronal-like phenotype and to form tubular/glomerular-like tridimensional structures. We the isolated the stem cell population form normal nephrospheres on the basis of the PKH fluorescence. We identified 3 PKH populations: PKHhigh population, with a high level of fluorescence, PKHlow population, with a low level of fluorescence, and PKHneg population, negative for PKH. The populations were separated with cell sorting and cultivated to form spheres. Only PKHhigh cells were able to generate new spheres, demostrating that the PKHhigh cells represent the stem cell population inside the nephrospheres. Normal and cancer PKHhigh cells will be deeply characterized in the future.
BIANCHI, CRISTINA
PEREGO, ROBERTO
renal cell carcinoma, kidney, stem cells, cancer stem cells, pkh, nephrosphere
MED/04 - PATOLOGIA GENERALE
Italian
Scuola di Dottorato in Scienze Mediche Sperimentali e Cliniche
TECNOLOGIE BIOMEDICHE - 20R
22
2008/2009
(2010). Isolamento e caratterizzazione di cellule staminali adulte da rene normale e carcinoma renale. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/7970
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