Serially Transplanted Nonpericytic CD146(-) Adipose Stromal/Stem Cells in Silk Bioscaffolds Regenerate Adipose Tissue In Vivo

Abstract

Progenitors derived from the stromal vascular fraction (SVF) of white adipose tissue (WAT) possess the ability to form clonal populations and differentiate along multiple lineage pathways. However, the literature continues to vacillate between defining adipocyte progenitors as "stromal" or "stem" cells. Recent studies have demonstrated that a nonpericytic subpopulation of adipose stromal cells, which possess the phenotype, CD45(-) /CD31(-) /CD146(-) /CD34(+) , are mesenchymal, and suggest this may be an endogenous progenitor subpopulation within adipose tissue. We hypothesized that an adipose progenitor could be sorted based on the expression of CD146, CD34, and/or CD29 and when implanted in vivo these cells can persist, proliferate, and regenerate a functional fat pad over serial transplants. SVF cells and culture expanded adipose stromal/stem cells (ASC) ubiquitously expressing the green fluorescent protein transgene (GFP-Tg) were fractionated by flow cytometry. Both freshly isolated SVF and culture expanded ASC were seeded in three-dimensional silk scaffolds, implanted subcutaneously in wild-type hosts, and serially transplanted. Six-week WAT constructs were removed and evaluated for the presence of GFP-Tg adipocytes and stem cells. Flow cytometry, quantitative polymerase chain reaction, and confocal microscopy demonstrated GFP-Tg cell persistence, proliferation, and expansion, respectively. Glycerol secretion and glucose uptake assays revealed GFP-Tg adipose was metabolically functional. Constructs seeded with GFP-Tg SVF cells or GFP-Tg ASC exhibited higher SVF yields from digested tissue, and higher construct weights, compared to nonseeded controls. Constructs derived from CD146(-) CD34(+) -enriched GFP-Tg ASC populations exhibited higher hemoglobin saturation, and higher frequency of GFP-Tg cells than unsorted or CD29(+) GFP-Tg ASC counterparts. These data demonstrated successful serial transplantation of nonpericytic adipose-derived progenitors that can reconstitute adipose tissue as a solid organ. These findings have the potential to provide new insights regarding the stem cell identity of adipose progenitor cells.

Keywords: Adipose stem cells; Adult stem cells; CD34+; Differentiation; Fluorescence-activated cell sorter; Progenitor cells; Stem cell transplantation; Stromal cells.

Figure 1. Characterization of cells used in the study

1a). Pie chart of subpopulations within GFP-Tg SVF cells that were utilized for SVF serial transplantation studies. 1b). Sorting and enrichment of CD146⁻ CD29⁺, and CD146⁻ CD34⁺ subpopulations that were culture expanded to P2 ASC for GFP-Tg ASC serial transplantation studies. 1c). Immunophenotype of GFP-Tg SVF cells and culture-expanded GFP-Tg ASC, until passage 2 (P2). All experiments were repeated in triplicate; sample size, n=3 per replicate. Values reported as mean ± standard deviation (µ ± SD); *p < 0.05, ** p < 0.01; ***p < 0.001.

Figure 2. Comparison of CD29-enriched and CD34-enriched GFP-Tg ASC

2a). Immunophenotype of CD146⁻ CD29⁺, and CD146⁻ CD34⁺ sorted GFP-Tg ASC subpopulations. Immunophenotypes were based on expression of CD29, CD31, CD34, CD45, CD11B, Sca-1, and CD105 surface antigens. 2c). Adipogenic differentiation, and 2d) colony formation assays of unfractionated GFP-Tg ASC, CD146⁻ CD29⁺ GFP-Tg ASC, and CD146⁻ CD34⁺ GFP-Tg ASC. All experiments were repeated in triplicate; sample size, n=3 per replicate. Values reported as mean ± standard deviation (µ ± SD); *p < 0.05, ** p < 0.01; ***p < 0.001.

Figure 3. Weekly progression of tissue engineered fat within and surrounding GFP-Tg SVF cell implants in mice

3a). Scaffolds were implanted with no cells, or with 500k GFP-Tg SVF cells in silk scaffolds. Scaffolds were removed following 1 week, 2 weeks, or 6 weeks of implantation. 3b) Percent hemoglobin saturation was measured immediately following implantation, and after 1 week and 6 weeks of initial implantation. 3c). Quantification of removed scaffold (explant) engraftment via ImageJ analyses of photomicrographs in 3a; sample size, n=6 per group. 3d). Scaffold mass measurements following weeks 1, 2, and 6 of implantation; sample size, n=20. Quantification supports photomicrographs of SVF-mediated engraftment acceleration after 1 week of implantation. 3e). Weekly SVF cell quantification correlate with SVF cell persistence and expansion by week 2, and differentiation. Values reported as mean ± standard deviation (µ ± SD); *p < 0.05, ** p < 0.01; ***p < 0.001.

Figure 4. Detection of GFP-Tg SVF from tissue engineered adipose using HFIP 6-week silk scaffold implants in mice over two serial transplantations

Photomicrographs of GFP-Tg SVF cell implantation following weeks 1, 4, and 6 in vivo demonstrate persistence, proliferation, and ability to form GFP-Tg adipose depots. 4a). Confocal microscopy images of GFP, BODIPY, DAPI, and merged images within initial 6-week GFP-Tg SVF cell (T₀) transplants. 4b) Merged GFP/BODIPY/DAPI images of removed 6-week GFP-Tg SVF cell first serial (T₁) and second serial (T₂) transplants; sample size, n=5. 4c) Flow cytometric analyses of GFP expression in removed adipose scaffolds following T₀, T₁, and T₂ transplants; sample size, n=18. 4d). ImageJ analysis of weekly confocal images of T₀ explants. 4e) Quantification of weekly flow cytometric analyses of GFP expression in removed T₀ GFP-Tg SVF cell implants. 4f). GFP DNA expression in samples from removed 6-week T₀, T₁, and T₂ transplants. GFP expression reported as fold expression and normalized to GAPDH expression. 4g). Immunophenotype based on expression of GFP, CD29, CD31, CD34, CD45, and Sca-1 antigen expression on SVF cells isolated from 6-week T₂ transplants. Values reported as mean ± standard deviation (µ ± SD); comparing individual grouping to GFP⁺ control: *p < 0.05, ** p < 0.01; ***p < 0.001; comparing to GFP-SVF T₂ seeded to unseeded control groups: ^& p < 0.05.

Figure 5. Adipose formation and functionality within GFP-Tg ASC seeded serial silk implants

5a). Photomicrographs of scaffolds that were implanted with no cells (control), or with (a), 500k unsorted GFP-Tg ASC; (b), CD146⁻ CD29⁺ GFP-Tg ASC; or (c), CD146⁻ CD34⁺ GFP-Tg ASC; in silk scaffolds. Scaffolds were removed following 6 weeks of implantation. 5b). Images of removed scaffolds from cohorts (a)-(c). 5c) Percent hemoglobin saturation was measured in groups (a)-(c) after 6 weeks of initial (T₀) implantation. 5d). Quantification of removed scaffold (explant) engraftment via ImageJ analyses of photomicrographs in 4b; sample size, n=6 per grouping. 5e). Scaffold mass measurements after 6-week implants of cohorts (a)-(c), and control implants that were not seeded with any cells. 5f). Quantification of SVF recovered from 6-week implants, and surrounding fat from cohorts (a)-(c), and the unseeded control group; sample size, n=18 per grouping. Functionality was measured via 5g) glucose uptake, and 5f) glycerol secretion assays using 6-week T₁ transplantation constructs. Data analyzed using Graphpad Prism. Two-way ANOVAs performed. Data reported as mean ± SD. *p < 0.05, ** p < 0.01; ***p < 0.001; comparing to CD34-enriched group: ^& p < 0.05.

Figure 6. Detection of CD146⁻ CD29⁺ GFP-Tg ASC and CD146⁻ CD34⁺ GFP-Tg ASC from tissue engineered adipose using HFIP 6-week silk scaffold implants in mice over two serial transplantations

Photomicrographs of GFP-Tg CD29-enriched and CD34-enriched (cohorts (a)-(c) cell implantation following week 6 in vivo demonstrate persistence, proliferation, and ability to form GFP-Tg adipose depots, similar to unsorted GFP-Tg SVF cohorts. 6a). Confocal microscopy images of GFP, BODIPY, DAPI, and merged images from 6-week GFP-Tg ASC serial (T₁) transplants. 6b) Confocal images of CD29-enriched and CD34-enriched 6-week T₁ groups reflected observation of micro vessel formation within CD34-enriched groups than in CD29-enriched groups; n=5 per group per replicate. Flow cytometric analyses of GFP expression in removed adipose scaffolds from cohorts (a)-(c) following 6c) T₀, and 6d) T₁ transplants. 6e). GFP DNA expression in samples from removed 6-week T₀ and T₁ transplants. GFP expression reported as fold expression and normalized to GAPDH expression. Quantification of %GFP positivity following 6-week transplantation in 6f) T₀ and 6g) T₁ GFP-Tg ASC cell implants. Data reported as mean + SD. Comparing individual grouping to unsorted cell seeded groups: *p < 0.05, ** p < 0.01; ***p < 0.001; comparing to CD34-enriched group: ^& p < 0.05

Figure 7. GFP-Tg SVF cells, CD29-enriched, and CD34-enriched GFP-Tg ASC infiltrate surrounding tissue to generate functional, GFP-Tg adipose

Confocal microscopic images of fat within 2mm surrounding SVF and ASC constructs revealed GFP positivity. 7a) Merged images of negative and positive control adipose stained with BODIPY and DAPI in non-GFP-Tg mice and GFP-Tg mice, respectively. 7b) Images of adipose surrounding 6-week constructs that were seeded with unsorted GFP-Tg SVF cells, unsorted GFP-Tg ASC, and CD146 CD29-enriched and CD34-enriched GFP-Tg ASC; n=5 per group per replicate. 7c) Quantification of confocal images from 7a and 7b. Functionality reported via 7d) glycerol secretion, and 7e) glucose uptake assays using 6-week T₁ transplantation constructs. Data reported as mean ± SD. *p < 0.05, ** p < 0.01; ***p < 0.001