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Prospective isolation of human bone marrow stromal cell subsets: A comparative study between Stro-1-, CD146- and CD105-enriched populations.

Gothard D, Greenhough J, Ralph E, Oreffo RO - J Tissue Eng (2014)

Bottom Line: This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1.The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations.However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies.

View Article: PubMed Central - PubMed

Affiliation: Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development and Health, Institute of Developmental Sciences, Southampton General Hospital, School of Medicine, University of Southampton, Southampton, UK.

ABSTRACT
Stro-1 has proved an efficacious marker for enrichment of skeletal stem and progenitor cells although isolated populations remain heterogeneous, exhibiting variable colony-forming efficiency and osteogenic differentiation potential. The emerging findings that skeletal stem cells originate from adventitial reticular cells have brought two further markers to the fore including CD146 and CD105 (both primarily endothelial and perivascular). This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1. Fluorescent cell sorting quantification showed that CD146 and CD105 both targeted smaller (2.22% ± 0.59% and 6.94% ± 1.34%, respectively) and potentially different human bone marrow stromal cell fractions compared to Stro-1 (16.29% ± 0.78%). CD146+, but not CD105+, cells exhibited similar alkaline phosphatase-positive colony-forming efficiency in vitro and collagen/proteoglycan deposition in vivo to Stro-1+ cells. Molecular analysis of a number of select osteogenic and potential osteo-predictive genes including ALP, CADM1, CLEC3B, DCN, LOXL4, OPN, POSTN and SATB2 showed Stro-1+ and CD146+ populations possessed similar expression profiles. A discrete human bone marrow stromal cell fraction (2.04% ± 0.41%) exhibited positive immuno-labelling for both Stro-1 and CD146. The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations. However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies.

No MeSH data available.


Related in: MedlinePlus

Gene expression analysis within dual-labelled populations. Cell populations were isolated via FACS from HBMSCs according to Stro-1/CD146, Stro-1/CD105 and CD146/CD105 expression. Unsorted HBMSCs were used as a control. Samples were cultured for 10 and 21 days in either basal or osteogenic media before rtPCR analysis for (a) ALP, (b) OPN, (c) CADM1, (d) CLEC3B, (e) DCN, (f) LOXL4, (g) POSTN and (h) SATB2 expression. (i) The immune regulatory gene, CTSC, was used as a negative control. Error bars are SD (*p ≤ 0.05, **p ≤ 0.01).MACS: magnetic-activated cell sorting; HBMSC: human bone marrow stromal cell; rtPCR: real-time polymerase chain reaction; SD: standard deviation.
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fig6-2041731414551763: Gene expression analysis within dual-labelled populations. Cell populations were isolated via FACS from HBMSCs according to Stro-1/CD146, Stro-1/CD105 and CD146/CD105 expression. Unsorted HBMSCs were used as a control. Samples were cultured for 10 and 21 days in either basal or osteogenic media before rtPCR analysis for (a) ALP, (b) OPN, (c) CADM1, (d) CLEC3B, (e) DCN, (f) LOXL4, (g) POSTN and (h) SATB2 expression. (i) The immune regulatory gene, CTSC, was used as a negative control. Error bars are SD (*p ≤ 0.05, **p ≤ 0.01).MACS: magnetic-activated cell sorting; HBMSC: human bone marrow stromal cell; rtPCR: real-time polymerase chain reaction; SD: standard deviation.

Mentions: Dual-labelled populations demonstrated a delayed response to osteoinduction with increased ALP expression visible at 21 days (Figure 6(a)). Stro-1-/CD146-enriched populations exhibited reduced CADM1 expression following 10 days of osteoinduction compared to unsorted populations (Figure 6(c)). Furthermore, only CD146-/CD105-enriched populations exhibited elevated CLEC3B expression in response to osteoinduction for 10 days (Figure 6(d)). No reproducible significant differences were observed between dual-labelled populations with and without osteoinduction in OPN, DCN, LOXL4, POSTN and SATB2 expression (Figure 6(b), (e)–(h), respectively). No significant differences in expression were observed in immune regulatory gene CTSC between populations with and without osteoinduction at day 21 (Figure 6(i)). However, higher CTSC expression in Stro-1-/CD146-enriched populations compared to Stro-1-/CD105- and CD146-/CD105-enriched populations after 10 days without osteoinduction was observed (Figure 6(i)). Data were derived from three patient samples.


Prospective isolation of human bone marrow stromal cell subsets: A comparative study between Stro-1-, CD146- and CD105-enriched populations.

Gothard D, Greenhough J, Ralph E, Oreffo RO - J Tissue Eng (2014)

Gene expression analysis within dual-labelled populations. Cell populations were isolated via FACS from HBMSCs according to Stro-1/CD146, Stro-1/CD105 and CD146/CD105 expression. Unsorted HBMSCs were used as a control. Samples were cultured for 10 and 21 days in either basal or osteogenic media before rtPCR analysis for (a) ALP, (b) OPN, (c) CADM1, (d) CLEC3B, (e) DCN, (f) LOXL4, (g) POSTN and (h) SATB2 expression. (i) The immune regulatory gene, CTSC, was used as a negative control. Error bars are SD (*p ≤ 0.05, **p ≤ 0.01).MACS: magnetic-activated cell sorting; HBMSC: human bone marrow stromal cell; rtPCR: real-time polymerase chain reaction; SD: standard deviation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4221949&req=5

fig6-2041731414551763: Gene expression analysis within dual-labelled populations. Cell populations were isolated via FACS from HBMSCs according to Stro-1/CD146, Stro-1/CD105 and CD146/CD105 expression. Unsorted HBMSCs were used as a control. Samples were cultured for 10 and 21 days in either basal or osteogenic media before rtPCR analysis for (a) ALP, (b) OPN, (c) CADM1, (d) CLEC3B, (e) DCN, (f) LOXL4, (g) POSTN and (h) SATB2 expression. (i) The immune regulatory gene, CTSC, was used as a negative control. Error bars are SD (*p ≤ 0.05, **p ≤ 0.01).MACS: magnetic-activated cell sorting; HBMSC: human bone marrow stromal cell; rtPCR: real-time polymerase chain reaction; SD: standard deviation.
Mentions: Dual-labelled populations demonstrated a delayed response to osteoinduction with increased ALP expression visible at 21 days (Figure 6(a)). Stro-1-/CD146-enriched populations exhibited reduced CADM1 expression following 10 days of osteoinduction compared to unsorted populations (Figure 6(c)). Furthermore, only CD146-/CD105-enriched populations exhibited elevated CLEC3B expression in response to osteoinduction for 10 days (Figure 6(d)). No reproducible significant differences were observed between dual-labelled populations with and without osteoinduction in OPN, DCN, LOXL4, POSTN and SATB2 expression (Figure 6(b), (e)–(h), respectively). No significant differences in expression were observed in immune regulatory gene CTSC between populations with and without osteoinduction at day 21 (Figure 6(i)). However, higher CTSC expression in Stro-1-/CD146-enriched populations compared to Stro-1-/CD105- and CD146-/CD105-enriched populations after 10 days without osteoinduction was observed (Figure 6(i)). Data were derived from three patient samples.

Bottom Line: This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1.The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations.However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies.

View Article: PubMed Central - PubMed

Affiliation: Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development and Health, Institute of Developmental Sciences, Southampton General Hospital, School of Medicine, University of Southampton, Southampton, UK.

ABSTRACT
Stro-1 has proved an efficacious marker for enrichment of skeletal stem and progenitor cells although isolated populations remain heterogeneous, exhibiting variable colony-forming efficiency and osteogenic differentiation potential. The emerging findings that skeletal stem cells originate from adventitial reticular cells have brought two further markers to the fore including CD146 and CD105 (both primarily endothelial and perivascular). This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1. Fluorescent cell sorting quantification showed that CD146 and CD105 both targeted smaller (2.22% ± 0.59% and 6.94% ± 1.34%, respectively) and potentially different human bone marrow stromal cell fractions compared to Stro-1 (16.29% ± 0.78%). CD146+, but not CD105+, cells exhibited similar alkaline phosphatase-positive colony-forming efficiency in vitro and collagen/proteoglycan deposition in vivo to Stro-1+ cells. Molecular analysis of a number of select osteogenic and potential osteo-predictive genes including ALP, CADM1, CLEC3B, DCN, LOXL4, OPN, POSTN and SATB2 showed Stro-1+ and CD146+ populations possessed similar expression profiles. A discrete human bone marrow stromal cell fraction (2.04% ± 0.41%) exhibited positive immuno-labelling for both Stro-1 and CD146. The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations. However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies.

No MeSH data available.


Related in: MedlinePlus