Human perivascular stem cell-derived extracellular vesicles mediate bone repair
- Johns Hopkins University, United States
- China Medical University, China
- UCLA, Orthopaedic Hospital, United States
- University of Edinburgh, United Kingdom
Figures
Figure 1 with 3 supplements
Perivascular stem cells (PSCs) promote BMSC proliferation, migration, and osteogenesis with extracellular vesicle (EV) elaboration in non-contact co-culture.
Human adipose tissue-derived perivascular stem cells (PSCs) were placed in transwell inserts and the effects on human BMSCs were assessed. (A) MTS assay among BMSCs after 72 hr with or without PSCs …
Figure 1—figure supplement 1
Human PSC derivation by fluorescence activated cell sorting.
From left to right: Perivascular stem cells are derived by size distribution, followed by exclusion of DAPI+ cells, followed by exclusion of CD31 or CD45 expressing cells. Finally, microvascular …
Figure 1—figure supplement 2
Flow cytometry analysis of FACS derived human PSC.
Human PSC, as defined by FACS purification from human lipoaspirate, were examined at passage eight by flow cytometry. Expression is shown in blue in relation to unstained control in red. No …
Figure 1—figure supplement 3
Flow cytometry analysis and multilineage differentiation potential of human culture-derived BMSC.
Human BMSC, as defined by culture-adherence to standard culture ware, were examined at passage two by (A,B) flow cytometry and (C–E) in vitro multilineage differentiation potential. (A,B) By flow …
Figure 2 with 3 supplements
PSC-EVs promote BMSC proliferation, migration, and mineralization.
(A) Appearance of BMSCs treated with or without PKH26 (red)-labeled PSC-EVs. (B) Appearance of BMSCs treated with or without pHrodo (red)-labeled PSC-EVs. Images after 48 hr, with DAPI nuclear …
Figure 2—figure supplement 1
PSC-EV promote ASC migration and mineralization.
In similarity to studies in human BMSC, human unpurified adipose-derived stem stromal cells (ASC) were incubated with PSC-EV at ascending dosages. (A) ASC proliferation assessed by MTS assay at 72 …
Figure 2—figure supplement 2
Comparative effects of human ASC-EVs and PSC-EVs on BMSC proliferation, migration, and osteogenesis.
(A) BMSC proliferation assessed by MTS assay at 72 hr with or without ASC-EVs and PSC-EVs (1–5 μg/mL). (B) BMSC migration assessed by scratch wound healing assay at 8 hr with or without ASC-EVs and …
Figure 2—figure supplement 3
Distribution of PSC-EV associated transcripts within control- or EV-treated BMSC.
(A) Identification of transcripts within PSC-EV. Minus versus average (MvA) plots of all transcripts in RNA sequencing of three PSC-EV samples. Blue dots indicate 7,789 PSC-EV transcripts with a …
Figure 3 with 1 supplement
PSC-EVs require tetraspanins for bioactive effects on BMSCs.
(A) Appearance of BMSCs treated with pHrodo (red)-labeled PSC-EVs in the context of incubation with neutralizing antibodies to CD9, CD81, or isotype control (IgG). Images after 48 hr, with DAPI …
Figure 3—figure supplement 1
PSC-EVs require surface-associated proteins for bioactive effects on BMSCs.
Trypsinization effect on PSC-EV bioactivity. PSC-EVs were pre-treated with trypsin, followed by re-isolation of EVs and application to BMSCs. (A) BMSC proliferation assessed by MTS assay at 72 hr …
Figure 4
PSC-EV bioactivity requires IGSF8 and PTGFRN expression on recipient BMSCs.
(A) Gene expression of either IGSF8 or PTGFRN as assessed by regular PCR after shRNA mediated knockdown (96 hr shown). (B) Appearance of IGSF8 or PTGFRN shRNA silenced BMSCs treated with pHrodo …
Figure 5 with 2 supplements
PSC-EVs promote calvarial bone regeneration in vivo.
PSC-EVs (1 or 2.5 μg) were percutaneously delivered twice weekly overlying a circular, full thickness frontal bone defect site (1.8 mm diameter). Analysis was performed at 4 weeks thereafter. See …
Figure 5—figure supplement 1
PSC-EVs promote mouse cell proliferation, migration, and mineralization.
Effects on PSC-EV were assayed using either mouse ASCs or neonatal mouse calvarial cells (NMCCs). (A) Mouse ASC and NMCC proliferation assessed by MTS assay at 72 hr with or without PSC-EVs (1–5 …
Figure 5—figure supplement 2
Animal treatment protocol.
Schematic representation of animal treatment protocol for in vivo study. A full-thickness frontal bone defect is created (1.8 mm diameter circular defect in the right frontal bone), followed by …
Additional files
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Supplementary file 1
Key Resources Table.
- https://doi.org/10.7554/eLife.48191.017
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Supplementary file 2
Frequency of human PSC in lipoaspirate used.
- https://doi.org/10.7554/eLife.48191.018
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Supplementary file 3
Yields of PSC-EV.
Yield produced by each PSC-EV isolation are summarized. Protein amounts of harvested PSC-EV were determined by the BCA method. For patient samples 1 and 2, the same cell population was used to isolate EVs at two different passages as indicated.
- https://doi.org/10.7554/eLife.48191.019
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Supplementary file 4
Highest 100 transcripts in human PSC-EVs.
- https://doi.org/10.7554/eLife.48191.020
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Supplementary file 5
Relative gene expression within human PSC-EVs or parent PSC among transcription factors enriched in porcine ASC-EVs.
- https://doi.org/10.7554/eLife.48191.021
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Supplementary file 6
Most upregulated pathways among PSC-EV-treated BMSC by Ingenuity Pathway Analysis.
- https://doi.org/10.7554/eLife.48191.022
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Supplementary file 7
Most downregulated pathways among PSC-EV-treated BMSC by Ingenuity Pathway Analysis.
- https://doi.org/10.7554/eLife.48191.023
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Supplementary file 8
CD markers enriched in PSC-EVs.
- https://doi.org/10.7554/eLife.48191.024
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Supplementary file 9
Animal allocation and treatment groups.
- https://doi.org/10.7554/eLife.48191.025
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Supplementary file 10
Antibodies used.
- https://doi.org/10.7554/eLife.48191.026
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Supplementary file 11
Quantitative PCR primers used.
- https://doi.org/10.7554/eLife.48191.027
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Supplementary file 12
Basic features of human PSCs, ASCs, and BMSCs.
- https://doi.org/10.7554/eLife.48191.028
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Transparent reporting form
- https://doi.org/10.7554/eLife.48191.029
