Age-Related Effects on the Potency of Human Adipose-Derived Stem Cells: Creation and Evaluation of Superlots and Implications for Musculoskeletal Tissue Engineering Applications

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed “superlots,” containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation

Primary Cilia: The Chemical Antenna Regulating Human Adipose-Derived Stem Cell Osteogenesis

Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries

Primary cilia: the chemical antenna regulating human adipose-derived stem cell osteogenesis.

Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries

Age-Related Effects on the Potency of Human Adipose-Derived Stem Cells: Creation and Evaluation of Superlots and Implications for Musculoskeletal Tissue Engineering Applications

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed “superlots,” containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation

Primary cilia on hASC.

<p>(a) The non-motile primary cilium axoneme is composed of 9 pairs of peripheral microtubule doublets arranged concentrically in 9+0 configuration, devoid of a central tubulin pair. (b–f) Primary cilia appear as a dense cluster of acetylated tubulin protruding from the apical surface of hASC in confluent culture. (b) Few cilia are observed 24 hours after culture in complete growth expansion media (CGM) in subconfluent culture. They are observed at day 3 (c) undifferentiated in CGM, (d) differentiated in osteogenic differentiation medium (ODM) and at day 12 in (e) undifferentiated in CGM and (f) differentiated in ODM. Cilia are identified with anti-acetylated tubulin (green) or in combination with CEP164 (red), actin identified with phalloidin (red) and nuclei are identified with DAPI staining (blue). Scale bar = 25 µm.</p

The effect of knockdown on hASC proliferation in expansion media (CGM).

<p>DNA was collected at days 0, 3, 7 and 10 to measure proliferative activity with siRNA knockdown of PC1, PC2 and IFT88 in as compared to the non-targeting scramble control siRNA. Knockdown of IFT88 significantly increases hASC proliferation as compared to control (n = 3, error bars = SEM, Student's t-test comparing each treatment to control, * p<0.05, ** p<0.01).</p

PC1, PC2 and IFT88 gene expression up to 15 days in ODM culture.

<p>siRNA knockdown applied at days 0 and 7, confers at least a 50% reduction in mRNA expression of each cilia-associate protein (n = 3, Error bars = SEM).</p

Calcium accretion is affected by cilia-associated protein knockdown.

<p>(a) Alizarin Red staining indicating degree of mineralization after 14 days ODM with knockdown. (b) Quantified calcium accretion normalized to DNA content. PC1 and PC2 knockdown leads to diminished calcium accretion, (n = 3, error bars = SEM, Student's t-test comparing each treatment to control, * p<0.05).</p

Osteogenic gene marker expression in hASC.

<p>IFT88 knockdown confers a reduction in (a) Runx2 gene expression after 3 day and (c) BMP-2 gene expression after 10 day culture in osteogenic differentiation medium (ODM). (b) PC1, PC2 and IFT88 knockdown confer a reduction in alkaline phosphatase (ALP) gene expression after 10 days of culture in ODM. (d) PC1 knockdown confers downregulation of osteocalcin after 15 days of culture in osteogenic differentiation. Knockdown treatments applied at day 0 and day 7 of differentiation, (n = 3, error bars = SEM, Student's t-test comparing each treatment to control, * p<0.05, ** p<0.01).</p