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. 2023 Jan-Dec:32:9636897231175968.
doi: 10.1177/09636897231175968.

Highly Pluripotent Adipose-Derived Stem Cell-Enriched Nanofat: A Novel Translational System in Stem Cell Therapy

Lindsey Alejandra Quintero Sierra  1 , Reetuparna Biswas  1 , Anita Conti  1 , Alice Busato  1   2 , Riccardo Ossanna  1 , Nicola Zingaretti  3 , Pier Camillo Parodi  3   4 , Giamaica Conti  1 , Michele Riccio  4   5 , Andrea Sbarbati  1   4 , Francesco De Francesco  5
Affiliations

Highly Pluripotent Adipose-Derived Stem Cell-Enriched Nanofat: A Novel Translational System in Stem Cell Therapy

Lindsey Alejandra Quintero Sierra et al. Cell Transplant. 2023 Jan-Dec.

Abstract

Fat graft is widely used in plastic and reconstructive surgery. The size of the injectable product, the unpredictable fat resorption rates, and subsequent adverse effects make it tricky to inject untreated fat into the dermal layer. Mechanical emulsification of fat tissue, which Tonnard introduced, solves these problems, and the product obtained was called nanofat. Nanofat is widely used in clinical and aesthetic settings to treat facial compartments, hypertrophic and atrophic scars, wrinkle attenuation, skin rejuvenation, and alopecia. Several studies demonstrate that the tissue regeneration effects of nanofat are attributable to its rich content of adipose-derived stem cells. This study aimed to characterize Hy-Tissue Nanofat product by investigating morphology, cellular yield, adipose-derived stem cell (ASC) proliferation rate and clonogenic capability, immunophenotyping, and differential potential. The percentage of SEEA3 and CD105 expression was also analyzed to establish the presence of multilineage-differentiating stress-enduring (MUSE) cell. Our results showed that the Hy-Tissue Nanofat kit could isolate 3.74 × 104 ± 1.31 × 104 proliferative nucleated cells for milliliter of the treated fat. Nanofat-derived ASC can grow in colonies and show high differentiation capacity into adipocytes, osteocytes, and chondrocytes. Moreover, immunophenotyping analysis revealed the expression of MUSE cell antigen, making this nanofat enriched of pluripotent stem cell, increasing its potential in regenerative medicine. The unique characteristics of MUSE cells give a simple, feasible strategy for treating a variety of diseases.

Keywords: adipose stem cells; mechanical disaggregation; nanofat; stem cell therapy; stromal vascular fraction; translational therapy.

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Conflict of interest statement

The study respects all ethical requirements in its targets and methodologies. We strictly comply with extensively identified global codes of practice inclusive of the Nuremberg Code, the Helsinki settlement, and the conventions of the Council of Europe on human rights and biomedicine, with a specific interest in European law: 2001/eighty-three/ec, 86/609/eec and fp7 selection nr 1982/2006ec. Human organic samples are essential due to the fact we want to test human cells, which have unique, organic traits distinct from the ones of animals. The general goal of this undertaking is to reduce the range of animal experiments. Adult patients who are capable of supplying consent became protected. All patients, which might be the topics of our observations, donated their consensus to scientific remedies and an eBook of their hospital situation and images. We have acquired written, informed consent from all patients. This was authorized by means of our inner moral committee (CARU—University of Verona), with authorization provided through human research committee number 2/2019.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Morphological analysis of Hy-Tissue Nanofat product. Representative images were selected among the eight treated samples. (A) Scheme of the Hy-Tissue Nanofat procedure; (B) light microscopy of Hy-Tissue Nanofat product obtained with the whole-mount method. The square (in Fig. 1B, left) indicates the location of the higher magnification (right) [scale bar: (left) 300 μm, (right) 100 μm]. The graph represents the size distribution of the lipid droplets. (C). Light microscopy of Hy-Tissue Nanofat product obtained with the whole-mount method after centrifugation. The square (in Fig. 1C, left) indicates the location of the higher magnification (Fig. 1C, right) [scale bar: (left) 80 μm, (right) 40 μm]. (D) Scanning electron microscopy of Hy-Tissue Nanofat product after centrifugation. The square (in Fig. 1D, left) indicates the location of the higher magnification (Fig. 1D, right); the elastic fiber, the connective tissue lamina, and the single collagen fiber are shown with a dotted arrow, an asterisk, and an arrow, respectively [scale bar: (left) 50 μm, (right) 5 μm].
Figure 2.
Figure 2.
Experimental methodology. The fat samples were each divided into three portions to be processed in N-ASC, N-ED-ASC and ED-ASC and evaluated for cell proliferation capacity, CFU-F assay, immunophenotyping and differentiation potential. N-ASC: nanofat-derived adipose-derived stem cell; N-ED-ASC: enzymatic digestion of nanofat adipose-derived stem cell; ED-ASC: collagenase-derived adipose-derived stem cell; CFU-F: colony-forming unit-fibroblast.
Figure 3.
Figure 3.
Cellular yield and proliferation capacity obtained with the three evaluated procedures of eight adipose tissue samples. (A) Nucleated cells obtained after the three evaluated treatments. Cell yield (no. of cell/ml FAT) was evaluated considering the enzymatic digestion as 100%. (B, left) Proliferation capacity of N-ASC, N-ED-ASC, and ED-ASC in T25 flasks. The days required for the adherent cells to reach confluence (passage 1) were counted. (B, right) The PDT of N-ASC, N-ED-ASC and ED-ASC was analyzed to evaluate the growth rate of adherent cells. No statistically significant differences were found between N-ASC and N-ED-ASC, and N-ED-ASC and ED-ASC. (C) Microscopic images of adherent cells 3 days after the extraction. All the results are shown as mean ± SD represented by error bars. Box and whisker plots represent the median. Significant statistical differences are indicated (*P < 0.05, **0.005 < P < 0.001, or ***P < 0.001). N-ASC: nanofat-derived adipose-derived stem cell; N-ED-ASC: enzymatic digestion of nanofat adipose-derived stem cell; ED-ASC: collagenase-derived adipose-derived stem cell; PDT: population doubling time assay.
Figure 4.
Figure 4.
Clonogenic potential of Hy-Tissue Nanofat product (n = 8). (A) Representative light microscope of CFU-F assay stained with toluidine blue (scale bar, 20 µm). (B) CFU-F yields of N-ASC, N-ED-ASC, and ED-ASC. (C) Percentage of CFU-F of N-ASC, N-ED-ASC, and ED-ASC. All the results are shown as mean ± SD represented by error bars. Significant statistical differences are indicated (*P < 0.05, **0.005 < P < 0.001, or ***P <0.001). CFU-F: colony-forming unit-fibroblast; N-ASC: nanofat-derived adipose-derived stem cell; N-ED-ASC: enzymatic digestion of nanofat adipose-derived stem cell; ED-ASC: collagenase-derived adipose-derived stem cell.
Figure 5.
Figure 5.
Surface markers expression was detected by flow cytometric analysis of N-ASC (n = 8). The percentage of positive cells for each marker was calculated after subtracting the non-specific fluorescence obtained with the control (unmarked). (A) Representative set of flow cytometry analysis for CD34, CD45, CD105, CD29, and CD73 markers performed on N-ASC. Percentage of positive cells to CD markers was indicated as an average of the samples; (B) percentage of positive cells to CD markers (as an average of the samples) in N-ASC compared with N-ED-ASC and ED-ASC; (C) percentage of positive cells to CD markers after in vitro cell expansion in N-ASC, N-ED-ASC, and ED-ASC. Results are presented as mean ± SD portrayed with error bars. Significant statistical differences are indicated (*P < 0.05). N-ASC: nanofat-derived adipose-derived stem cell; N-ED-ASC: enzymatic digestion of nanofat adipose-derived stem cell; ED-ASC: collagenase-derived adipose-derived stem cell.
Figure 6.
Figure 6.
Multilineage differentiation assay (n = 8). (A) Optical microscopy images of induced N-ASC, N-ED-ASC, and ED-ASC and non-induced (CTR) cells with differentiation medium at day 4 (Oil-Red O staining scale bar, 10 μm; Alzarin red staining scale bar, 20 μm; Alcian blue staining scale bar, 10 μm); (B) optical microscopy images of induced N-ASC, N-ED-ASC, and ED-ASC and non-induced (CTR) cells with differentiation medium at day 9 (Oil-Red O staining scale bar 10 μm; Alzarin red staining scale bar 20 μm; Alcian blue staining scale bar, 10 μm). Red spots indicate the accumulation of neutral lipid vacuoles stained with Oil-Red-Oil; Alzarin red staining reveals in red the extracellular matrix calcification; deposition of sulfated proteoglycan-rich matrix is marked in blue with Alcian blue staining; (C) graph represents the mean amount of lipid droplets of the induced ASC. After 9 days of the induction, N-ASC shows higher lipid droplet formation than ED-ASC, N-ED-ASC, and CTR. (D) The graph showed the extracellular matrix calcification area measurement (µm²) of the induced ASC. After 9 days of treatment, N-ASC showed the highest calcium deposit formation. (E) The graph represents the area measurement of the generated cartilage-like matrix (µm²). On the ninth day, N-ASC showed the larger cartilage deposit formation. In graphics C, D and E, the pink line represents N-ASC, the green line ED-ASC, and the blue line N-ED-ASC. The data are presented as mean ± SD; significant statistical differences are indicated (*P < 0.05, **0.05 < P < 0.001, ***P < 0.001 or ****P < 0.0001). N-ASC: nanofat-derived adipose-derived stem cell; N-ED-ASC: enzymatic digestion of nanofat adipose-derived stem cell; ED-ASC: collagenase-derived adipose-derived stem cell; CTR: control.
Figure 7.
Figure 7.
MUSE cell expression (n = 8). (A) Flow cytometry of N-ASC, N-ED-ASC, and ED-ASC to investigate the presence of MUSE cells. The percentage of positive cells to SEEA-3 and CD105 markers was indicated as an average of three samples, and the results are presented as mean ± SD. (B) Immunofluorescence microscopy of MUSE cells. MUSE cells in N-ASC were detected as positive cells for CD105 (left), SEEA3 (middle), and the simultaneous expression of CD105-SEEA3 (right). N-ASC: nanofat-derived adipose-derived stem cell; N-ED-ASC: enzymatic digestion of nanofat adipose-derived stem cell; ED-ASC: collagenase-derived adipose-derived stem cell; MUSE: multilineage-differentiating stress-enduring.
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