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Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1.

Kluth SM, Radke TF, Kögler G - Stem Cells Int (2013)

Bottom Line: Multipotent stromal cells can be isolated from a variety of different tissues in the body.In this study, experiments assessing the haematopoiesis-supporting capacity and molecular biological analyses were conducted and clearly confirmed different properties.Compared to CB MSC, USSC lead to a higher expansion of haematopoietic cells and in addition express significantly higher levels of insulin-like growth factor binding protein 1 (IGFBP1), but lower levels of IGF2.

View Article: PubMed Central - PubMed

Affiliation: Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, 40225 Duesseldorf, Germany.

ABSTRACT
Multipotent stromal cells can be isolated from a variety of different tissues in the body. In contrast to stromal cells from the adult bone marrow (BM) or adipose tissue, cord blood (CB) multipotent stromal cells (MSC) are biologically younger. Since first being described by our group, delta like 1 homologue (DLK-1) was determined as a discriminating factor between the distinct cord blood-derived subpopulations: the unrestricted somatic stromal cells (USSC), which lack adipogenic differentiation capacity, and the BM MSC-like CB MSC. In this study, experiments assessing the haematopoiesis-supporting capacity and molecular biological analyses were conducted and clearly confirmed different properties. Compared to CB MSC, USSC lead to a higher expansion of haematopoietic cells and in addition express significantly higher levels of insulin-like growth factor binding protein 1 (IGFBP1), but lower levels of IGF2. The data presented here also indicate that DLK-1 might not be the sole factor responsible for the inhibition of adipogenic differentiation potential in USSC but nevertheless indicates a biological diversity among cord blood-derived stromal cells.

No MeSH data available.


Related in: MedlinePlus

DLK-1 in USSC: inhibitor of adipogenesis? (a) Representative immunofluorescence staining of CB MSC (n = 3), USSC (n = 4) and HepG2. USSC revealed a clear DLK-1 expression, detected by immunofluorescence, while CB MSC were completely negative. The hepatocarcinoma cell line HepG2 served as biological positive control. (b) DLK-1 flowcytometry analysis was applied to clearly distinguish between intracellular and extracellular expression of DLK-1 protein. No DLK-1 expression could be determined in USSC by extracellular staining. After permeabilization a small DLK-1 positive subpopulation was detected in the USSC lines by intracellular staining. DLK-1 overexpressing CB MSC were used a positive control for extracellular staining. (c) By ectodomain shedding, the soluble and adipogenic inhibitory DLK-1 protein is cleaved. To validate whether USSC express the functional DLK-1, an ELISA was performed. The supernatant of 3 days standard cultures of USSC (n = 4) and CB MSC (n = 3) was analyzed, maternal plasma was used as positive control. No specific DLK-1 release was detected in any of the analyzed USSC or CB MSC, questioning function of DLK-1 expression in USSC. However, a clear secretion of DLK-1 was detected in the CB MSC overexpressing DLK-1. (d) A heterogeneous expression of CEBPα (CB MSC n = 4, CB MSC-derived clones n = 3, USSC n = 2, USSC-derived clones n = 2) could be detected in different USSC and CB MSC cell lines analyzed by real time PCR analysis. (e) Real time PCR analysis revealed a heterogenous expression of PPARγ2 expression (CB MSC n = 3, USSC n = 4) in the USSC and CB MSC cell lines.
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fig2: DLK-1 in USSC: inhibitor of adipogenesis? (a) Representative immunofluorescence staining of CB MSC (n = 3), USSC (n = 4) and HepG2. USSC revealed a clear DLK-1 expression, detected by immunofluorescence, while CB MSC were completely negative. The hepatocarcinoma cell line HepG2 served as biological positive control. (b) DLK-1 flowcytometry analysis was applied to clearly distinguish between intracellular and extracellular expression of DLK-1 protein. No DLK-1 expression could be determined in USSC by extracellular staining. After permeabilization a small DLK-1 positive subpopulation was detected in the USSC lines by intracellular staining. DLK-1 overexpressing CB MSC were used a positive control for extracellular staining. (c) By ectodomain shedding, the soluble and adipogenic inhibitory DLK-1 protein is cleaved. To validate whether USSC express the functional DLK-1, an ELISA was performed. The supernatant of 3 days standard cultures of USSC (n = 4) and CB MSC (n = 3) was analyzed, maternal plasma was used as positive control. No specific DLK-1 release was detected in any of the analyzed USSC or CB MSC, questioning function of DLK-1 expression in USSC. However, a clear secretion of DLK-1 was detected in the CB MSC overexpressing DLK-1. (d) A heterogeneous expression of CEBPα (CB MSC n = 4, CB MSC-derived clones n = 3, USSC n = 2, USSC-derived clones n = 2) could be detected in different USSC and CB MSC cell lines analyzed by real time PCR analysis. (e) Real time PCR analysis revealed a heterogenous expression of PPARγ2 expression (CB MSC n = 3, USSC n = 4) in the USSC and CB MSC cell lines.

Mentions: To analyze the DLK-1 protein level in USSC, immunofluorescence assays were performed. USSC (n = 4) exhibited a DLK-1 protein expression, while CB MSC (n = 3) were DLK-1 negative (Figure 2(a)) as described before [3]. In order to validate these data, the DLK-1 expression was additionally analyzed by flow cytometry applying a monoclonal antibody detecting the extracellular domain of DLK-1/FA1. Surprisingly, no extracellular expression of DLK-1 protein in USSC could be verified (Figure 2(b)), yet in some of the USSC lines examined, intracellular antibody labeling revealed a small but clearly DLK-1-positive subpopulation of USSC, suggesting intracellular localization of DLK-1/FA1.


Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1.

Kluth SM, Radke TF, Kögler G - Stem Cells Int (2013)

DLK-1 in USSC: inhibitor of adipogenesis? (a) Representative immunofluorescence staining of CB MSC (n = 3), USSC (n = 4) and HepG2. USSC revealed a clear DLK-1 expression, detected by immunofluorescence, while CB MSC were completely negative. The hepatocarcinoma cell line HepG2 served as biological positive control. (b) DLK-1 flowcytometry analysis was applied to clearly distinguish between intracellular and extracellular expression of DLK-1 protein. No DLK-1 expression could be determined in USSC by extracellular staining. After permeabilization a small DLK-1 positive subpopulation was detected in the USSC lines by intracellular staining. DLK-1 overexpressing CB MSC were used a positive control for extracellular staining. (c) By ectodomain shedding, the soluble and adipogenic inhibitory DLK-1 protein is cleaved. To validate whether USSC express the functional DLK-1, an ELISA was performed. The supernatant of 3 days standard cultures of USSC (n = 4) and CB MSC (n = 3) was analyzed, maternal plasma was used as positive control. No specific DLK-1 release was detected in any of the analyzed USSC or CB MSC, questioning function of DLK-1 expression in USSC. However, a clear secretion of DLK-1 was detected in the CB MSC overexpressing DLK-1. (d) A heterogeneous expression of CEBPα (CB MSC n = 4, CB MSC-derived clones n = 3, USSC n = 2, USSC-derived clones n = 2) could be detected in different USSC and CB MSC cell lines analyzed by real time PCR analysis. (e) Real time PCR analysis revealed a heterogenous expression of PPARγ2 expression (CB MSC n = 3, USSC n = 4) in the USSC and CB MSC cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: DLK-1 in USSC: inhibitor of adipogenesis? (a) Representative immunofluorescence staining of CB MSC (n = 3), USSC (n = 4) and HepG2. USSC revealed a clear DLK-1 expression, detected by immunofluorescence, while CB MSC were completely negative. The hepatocarcinoma cell line HepG2 served as biological positive control. (b) DLK-1 flowcytometry analysis was applied to clearly distinguish between intracellular and extracellular expression of DLK-1 protein. No DLK-1 expression could be determined in USSC by extracellular staining. After permeabilization a small DLK-1 positive subpopulation was detected in the USSC lines by intracellular staining. DLK-1 overexpressing CB MSC were used a positive control for extracellular staining. (c) By ectodomain shedding, the soluble and adipogenic inhibitory DLK-1 protein is cleaved. To validate whether USSC express the functional DLK-1, an ELISA was performed. The supernatant of 3 days standard cultures of USSC (n = 4) and CB MSC (n = 3) was analyzed, maternal plasma was used as positive control. No specific DLK-1 release was detected in any of the analyzed USSC or CB MSC, questioning function of DLK-1 expression in USSC. However, a clear secretion of DLK-1 was detected in the CB MSC overexpressing DLK-1. (d) A heterogeneous expression of CEBPα (CB MSC n = 4, CB MSC-derived clones n = 3, USSC n = 2, USSC-derived clones n = 2) could be detected in different USSC and CB MSC cell lines analyzed by real time PCR analysis. (e) Real time PCR analysis revealed a heterogenous expression of PPARγ2 expression (CB MSC n = 3, USSC n = 4) in the USSC and CB MSC cell lines.
Mentions: To analyze the DLK-1 protein level in USSC, immunofluorescence assays were performed. USSC (n = 4) exhibited a DLK-1 protein expression, while CB MSC (n = 3) were DLK-1 negative (Figure 2(a)) as described before [3]. In order to validate these data, the DLK-1 expression was additionally analyzed by flow cytometry applying a monoclonal antibody detecting the extracellular domain of DLK-1/FA1. Surprisingly, no extracellular expression of DLK-1 protein in USSC could be verified (Figure 2(b)), yet in some of the USSC lines examined, intracellular antibody labeling revealed a small but clearly DLK-1-positive subpopulation of USSC, suggesting intracellular localization of DLK-1/FA1.

Bottom Line: Multipotent stromal cells can be isolated from a variety of different tissues in the body.In this study, experiments assessing the haematopoiesis-supporting capacity and molecular biological analyses were conducted and clearly confirmed different properties.Compared to CB MSC, USSC lead to a higher expansion of haematopoietic cells and in addition express significantly higher levels of insulin-like growth factor binding protein 1 (IGFBP1), but lower levels of IGF2.

View Article: PubMed Central - PubMed

Affiliation: Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, 40225 Duesseldorf, Germany.

ABSTRACT
Multipotent stromal cells can be isolated from a variety of different tissues in the body. In contrast to stromal cells from the adult bone marrow (BM) or adipose tissue, cord blood (CB) multipotent stromal cells (MSC) are biologically younger. Since first being described by our group, delta like 1 homologue (DLK-1) was determined as a discriminating factor between the distinct cord blood-derived subpopulations: the unrestricted somatic stromal cells (USSC), which lack adipogenic differentiation capacity, and the BM MSC-like CB MSC. In this study, experiments assessing the haematopoiesis-supporting capacity and molecular biological analyses were conducted and clearly confirmed different properties. Compared to CB MSC, USSC lead to a higher expansion of haematopoietic cells and in addition express significantly higher levels of insulin-like growth factor binding protein 1 (IGFBP1), but lower levels of IGF2. The data presented here also indicate that DLK-1 might not be the sole factor responsible for the inhibition of adipogenic differentiation potential in USSC but nevertheless indicates a biological diversity among cord blood-derived stromal cells.

No MeSH data available.


Related in: MedlinePlus