Growth and Differentiation of Human Adipose Stromal Cells in Culture

Strutt, Brenda; Khalil, Wahid; Killinger, D.W.

doi:10.1385/0-89603-335-x:41

Cited by 12 publications

(12 citation statements)

References 7 publications

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“…The methods used in our study (isolation, in vitro stimulated differentiation, and Oil Red O staining of human primary preadipocytes) are based on well-established protocols (33). As shown by Ramírez-Zacarías et al (30), quantitation of lipid accumulation by Oil Red O staining correlates well with the measurement of glycerophosphate dehydrogenase activity.…”

Section: Discussionmentioning

confidence: 83%

Mature Adipocytes Inhibit In Vitro Differentiation of Human Preadipocytes via Angiotensin Type 1 Receptors

et al. 2002

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Recent studies suggest that angiotensin II (Ang II) plays a role in the adipogenesis of murine preadipocytes. Here, we examined the role of Ang II for the differentiation of primary cultured human preadipocytes. Preadipocytes were isolated from human adipose tissue and stimulated to differentiate. Quantitation of gene expression during adipogenesis was performed for renin-angiotensin system (RAS) genes. The influence of the RAS on adipogenic differentiation was investigated by addition of either angiotensinogen (AGT), Ang II, or angiotensin receptor antagonists to the differentiation medium. We also examined the influence of adipocytes on adipogenesis by co-culture experiments. Expression of the RAS genes AGT, renin, angiotensin-converting enzyme, and Ang II type 1 receptor increased during adipogenesis. Stimulation of the Ang II type 1 receptor by Ang II reduced adipose conversion, whereas blockade of this receptor markedly enhanced adipogenesis. Adipocytes were able to inhibit preadipocyte differentiation in the co-culture, and this effect was abolished by blockade of the Ang II type 1 receptor. This finding points to a functional role of the RAS in the differentiation of human adipose tissue. Because AGT secretion and Ang II generation are characteristic features of adipogenesis, we postulate a paracrine negative-feedback loop that inhibits further recruitment of preadipocytes by maturing adipocytes. Diabetes 51: 1699 -1707, 2002 O besity is well recognized as a major risk factor for the development of type 2 diabetes. Paradoxically, loss of adipose tissue (1) or impaired adipogenic differentiation (2) have been proposed as important mechanisms underlying the development of insulin resistance and type 2 diabetes (lipotoxicity hypothesis [3]). Adipogenesis results from a precise interplay of transcription factors, enabling preadipocytes to accumulate lipids and respond to insulin (4). Interestingly, mature adipocytes secrete a host of vasoactive substances that influence adipogenesis (5), including the adipogenic vasodilators nitric oxide (6) and prostacyclin (7) and the antiadipogenic vasoconstrictor endothelin-1 (8), suggesting a paracrine role of vasoactive molecules in the regulation of adipocyte growth and differentiation.Angiotensin II (Ang II), another vasoactive molecule, has also recently been implicated in the modulation of adipogenesis (9). Thus, mature adipocytes express all components of the renin-angiotensin system (RAS), including angiotensinogen (AGT), the sole precursor of Ang II, as well as the angiotensin peptide forming enzymes renin, ACE, and chymase. Adipocytes also express the type 1 (AT 1 ) and type 2 (AT 2 ) angiotensin receptor subtypes (10). AGT expression and secretion increases during adipogenesis (11)(12)(13)(14), whereas suppression of the differentiation-specific element binding protein, the activator of differentiation-dependent AGT gene expression, suppressed AGT expression and adipogenesis in 3T3-L1 mouse clonal preadipocytes (15). Although previous studies in rodents poi...

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Section: Discussionmentioning

confidence: 83%

Mature Adipocytes Inhibit In Vitro Differentiation of Human Preadipocytes via Angiotensin Type 1 Receptors

et al. 2002

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“…Whereas the nonpolar fluorescein diacetate can cross the cell membrane, metabolically active cells can hydrolyze the ester to release the polar fluorescein molecule, which is not membrane permeable, leading to accumulation in viable cells. Because cell viability stains are very difficult to quantitate on clumps of adipose tissue, we used a standard collagenase digestion technique to dissociate the recovered adipose tissue 16. Only when cells are disassociated can one accurately estimate the percentage of cell survival from a particular processing method without digital image analysis.…”

Section: Discussionmentioning

confidence: 99%

Cryopreservation of Human Fat for Soft Tissue Augmentation

Moscatello¹,

Dougherty²,

Narins³

et al. 2005

Dermatologic Surgery

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Viability of adipocytes frozen at -20 degrees C was very low when analyzed by staining, and no cultures could be established from any of the specimens. In contrast, viable adipocytes were recovered from samples that were controlled-rate frozen in the presence of cryoprotectants and stored in nitrogen vapor. CONCLUSION. Our results indicate that fat frozen at -20 degrees C is not viable and thus provides no advantage over inert fillers. The methods here described could readily be transferred to the clinical setting after further laboratory study.

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“…Critical Step: hASCs can be passaged and expanded using standard cell culture techniques [26]. Our lab uses TrypLE Express reagent for digestion and split ratios of 1:4 every 3–5 days.…”

Section: Methodsmentioning

confidence: 99%

Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

et al. 2010

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Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques utilize integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. Here we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thereby capable of high expression in mammalian cells. Their repeated transfection into hASCs, an abundant somatic cell source that is amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ~4 weeks.

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Growth and Differentiation of Human Adipose Stromal Cells in Culture

Cited by 12 publications

References 7 publications

Mature Adipocytes Inhibit In Vitro Differentiation of Human Preadipocytes via Angiotensin Type 1 Receptors

Mature Adipocytes Inhibit In Vitro Differentiation of Human Preadipocytes via Angiotensin Type 1 Receptors

Cryopreservation of Human Fat for Soft Tissue Augmentation

Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

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