High plasticity of pediatric adipose tissue-derived stem cells: too much for selective skeletogenic differentiation?

Abstract

Stem cells derived from adipose tissue are a potentially important source for autologous cell therapy and disease modeling, given fat tissue accessibility and abundance. Critical to developing standard protocols for therapeutic use is a thorough understanding of their potential, and whether this is consistent among individuals, hence, could be generally inferred. Such information is still lacking, particularly in children. To address these issues, we have used different methods to establish stem cells from adipose tissue (adipose-derived stem cells [ADSCs], adipose explant dedifferentiated stem cells [AEDSCs]) from several pediatric patients and investigated their phenotype and differentiation potential using monolayer and micromass cultures. We have also addressed the overlooked issue of selective induction of cartilage differentiation. ADSCs/AEDSCs from different patients showed a remarkably similar behavior. Pluripotency markers were detected in these cells, consistent with ease of reprogramming to induced pluripotent stem cells. Significantly, most ADSCs expressed markers of tissue-specific commitment/differentiation, including skeletogenic and neural markers, while maintaining a proliferative, undifferentiated morphology. Exposure to chondrogenic, osteogenic, adipogenic, or neurogenic conditions resulted in morphological differentiation and tissue-specific marker upregulation. These findings suggest that the ADSC "lineage-mixed" phenotype underlies their significant plasticity, which is much higher than that of chondroblasts we studied in parallel. Finally, whereas selective ADSC osteogenic differentiation was observed, chondrogenic induction always resulted in both cartilage and bone formation when a commercial chondrogenic medium was used; however, chondrogenic induction with a transforming growth factor β1-containing medium selectively resulted in cartilage formation. This clearly indicates that careful simultaneous assessment of bone and cartilage differentiation is essential when bioengineering stem cell-derived cartilage for clinical intervention.

Figure 1.

Phase images of ADSC and adipose explant dedifferentiated stem cell cultures. (A): Diagram indicating the two approaches used to obtain adipose tissue-derived stem cells from pediatric abdominal fat and summary of donors' conditions (details can be found in supplemental online Table 1). (B): ADSC morphology at passage 1. (C): ADSC morphology at passage 4. Note the appearance of cells with a more spread out morphology and increased cytoplasm/nucleus ratio (arrows). (D): Adipose tissue explant from which cells are migrating. Cells have a multilocular morphology close to the explant (arrows) and become fibroblastic as they migrate away from it. (E, F): Expression of the cell surface proteins CD44, CD90, and CD105 assessed by flow cytometry in ADSCs (E) and chondroblasts (F) from the same patient (red curves: negative controls; blue curves: labeled cells). Scale bar = 200 μm. Abbreviations: ADSC, adipose-derived stem cell; FITC, fluorescein isothiocyanate; max, maximum; PE, phycoerythrin.

Figure 2.

Protein expression in adipose-derived stem cells detected by immunocytochemistry. (A): Vimentin. (B): Nestin. (C): Merged image of (A) and (B). (D): CD44. (E): Acetylated tubulin. (F): Aquaporin-1. (G): p75NTR. (H): Tgfβ1. (I): VEGF. (J): A2B5. (K): NF-200. (L): βIII-tubulin. (M): Gfap. (N, O): Controls for Alexa 568 anti-mouse (N) and Alexa 488 anti-rabbit (O) immunoglobulin staining (no primary antibody). Nuclei are visualized with 4′,6-diamidino-2-phenylindole (blue). Scale bars = 50 μm (D, L) and 100 μm for all other panels. Abbreviations: Gfap, glial fibrillary acidic protein; NF-200, neurofilament-200; Tgfβ1, transforming growth factor β1; VEGF, vascular endothelial growth factor.

Figure 3.

Expression of pluripotency markers in ADSCs and induced pluripotent stem cell generation. (A): Expression of pluripotency markers in AEDSCs and ADSCs in different patients (h6 and h2). hES cells were used as positive controls. (B): ADSCs plated on Matrigel-coated plates. (C, D): Morphological changes and rearrangement of cells 10 days post-lentiviral vector infection (C) and after replating (D). (E, F): Expression of SSEA3 and SSEA4, respectively. (G): Negative control (no primary antibody) for immunocytochemistry. Scale bars = 150 μm (B, C, E–G) and 400 μm (D). Abbreviations: ADSC, adipose-derived stem cell; AEDSC, adipose explant dedifferentiated stem cell; hES, human embryonic stem; Neg. con, negative control; SSEA, stage-specific embryonic antigen.

Figure 4.

Adipogenic, osteogenic, chondrogenic, and neurogenic differentiation of pediatric adipose-derived stem cells (ADSCs). (A): Reverse transcription-polymerase chain reaction showing de novo expression of LPL and PPARγ upon adipogenic differentiation, de novo expression of ALP and OC and upregulation of OX upon osteogenic differentiation, and de novo expression of COLII and upregulation of RUNX2 and AG upon chondrogenic differentiation. (B): Cells stained with Oil Red O. (C): Cells stained with alizarin red. (D, E): Alcian Blue staining of cultures differentiated in either Tgfβ1-containing medium (C) or StemPro-C (D). (Dd, Ee): Osteogenesis detected by alizarin red staining in StemPro-C (E), but not in Tgfβ1-containing medium (D) treated cells. (F–H): COLII staining in ADSCs treated with Tgfβ1-containing medium (F) and StemPro-C (G) and in a section of human costal cartilage (H). (Ff): COLII expression in uninduced ADSCs. (Gg): Negative control for COLII staining of ADSCs (no primary antibody). (I): Negative control for COLII staining of costal cartilage (no primary antibody). (J): Quantification of chondrogenesis (Alcian Blue quantification). Data are expressed as fold differences, taking untreated controls as 1; ∗∗, p < .001. (K): Quantification of osteogenesis (alizarin red quantification). Data are expressed as fold differences, taking untreated controls as 1; ∗∗, p < .001. (L): Expression of neural differentiation markers after 2 weeks in control or neurogenic medium (B–E). Fetal brain cDNA was used as a positive control. (M): Phase image of control cells. (N–P): Phase images of cells grown in neurogenic medium for 2 weeks. Scale bars = 300 μm (B) ([C–G] are at the same magnification), 100 μm (H) ([H, I] are at the same magnification), and 50 μm (M–P). Abbreviations: AG, aggrecan; ALP, alkaline phosphatase; COLII, collagen II; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; LPL, lipoprotein lipase; neg, negative control; NF-200, neurofilament-200; NSE, neuron-specific enolase; OC, osteocalcin; OX, Osterix; PPARγ, peroxisome proliferator-activated receptorγ; Tgfβ1, transforming growth factor β1.

Figure 5.

Adipogenic, chondrogenic, and osteogenic differentiation of chondroblasts from costal cartilage. (A): Chondroblasts migrating out of the costal cartilage explant at 4 days. (B): Chondroblasts migrating out of the explant at 10 days. (C): Reverse transcription-polymerase chain reaction showing upregulation of COLII, AG, and RUNX2 upon chondrogenic differentiation, upregulation of ALP and de novo expression of OX upon osteogenic differentiation, and de novo expression of LPL and PPARγ upon adipogenic differentiation. (D, E): Oil Red O staining of chondroblasts treated for 3 weeks with control medium (D) or adipogenic medium (E). (F, G): Uninduced cells stained with Alcian Blue (F) and alizarin red (G). (H, I): Tgfβ1-containing medium-treated cells stained with Alcian Blue (H) and alizarin red (I). (J, K): StemPro-C-treated cells stained with Alcian Blue (J) and alizarin red (K). (L, M): Osteogenic medium-treated cells stained with Alcian Blue (L) and alizarin red (M). (N): Quantification of chondrogenesis (Alcian Blue quantification). Data are expressed as fold differences, taking untreated controls as 1; ∗, p < .002. (O): Quantification of osteogenesis (alizarin red quantification). Data are expressed as fold differences, taking untreated controls as 1. Scale bars = 300 μm (A, B, L) ([F–M] are at the same magnification) and 50 μm (E) ([D, E] are at the same magnification). Abbreviations: AB, Alcian Blue; AG, aggrecan; AR, alizarin red; ALP, alkaline phosphatase; COLII, collagen II; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LPL, lipoprotein lipase; osteog, osteogenic; OX, Osterix; PPARγ, peroxisome proliferator-activated receptor γ; Tgfβ1, transforming growth factor β1.

Figure 6.

Chondrogenic and osteogenic differentiation of pediatric ADSC and chondroblast micromass cultures in Tgfβ1-containing medium and StemPro-C assessed by histochemistry and Q-PCR. (A): Alcian Blue, alizarin red, and collagen II staining of ADSC and chondroblast micromass sections. Scale bars = 250 μm. (B): Q-PCR analysis of differentiation markers. The y-axes indicate fold changes in relation to untreated chondroblasts. Each experiment was done in triplicate or quadruplicate, and the results shown are representative of those obtained from three patients. Note significant osteocalcin upregulation in ADSC micromass cultures differentiated in StemPro-C; ∗, p < .05; ∗∗, p < .001. Abbreviations: ADSC, adipose-derived stem cell; ALP, alkaline phosphatase; IHH, Indian hedgehog; Q-PCR, quantitative polymerase chain reaction; Tgfβ1, transforming growth factor β1.