Lauren E. Kokai

Adipose stem cells: biology and clinical applications for tissue repair and regeneration.

There is a clear clinical need for cell therapies to repair or regenerate tissue lost to disease or trauma. Adipose tissue is a renewable source of stem cells, called adipose-derived stem cells (ASCs), that release important growth factors for wound healing, modulate the immune system, decrease inflammation, and home in on injured tissues. Therefore, ASCs may offer great clinical utility in regenerative therapies for afflictions such as Parkinsons disease and Alzheimers disease, spinal cord injury, heart disease, and rheumatoid arthritis, or for replacing lost tissue from trauma or tumor removal. This article discusses the regenerative properties of ASCs that can be harnessed for clinical applications, and explores current and future challenges for ASC clinical use. Such challenges include knowledge-based deficiencies, hurdles for translating research to the clinic, and barriers to establishing a new paradigm of medical care. Clinical experience with ASCs, ASCs as a portion of the heterogeneous stromal cell population extracted enzymatically from adipose tissue, and stromal vascular fraction are also described.

The potential of adipose-derived adult stem cells as a source of neuronal progenitor cells.

Summary: Adipose-derived adult stem cells are a population of mesenchymal stem cells extracted from discarded adipose tissue. Many have reported the differentiation of adipose-derived stem cells into chondrocytes, myocytes, osteoblasts, and, most recently, neural progenitor cells. This article covers the current state of the potential of the differentiation of adipose-derived stem cells into neuronal cells and an overview of their potential as adult stem therapies for neurological disorders. It has been reported that adipose-derived stem cells are capable of undergoing neuronal differentiation using protocols similar to that of Woodbury et al., which reported the differentiation of bone marrow stromal cells specifically into neurons. However, the transdifferentiation of bone marrow stromal cells into neuronal cells has recently fallen under intense criticism, which will likely place the plasticity of adipose-derived stem cells under scrutiny as well. To date, no group has produced evidence that adipose-derived stem cells are capable of differentiating to mature, functional neuronal cells in vitro. However, recent in vivo studies with adipose-derived stem cells are promising.

Incorporation of double-walled microspheres into polymer nerve guides for the sustained delivery of glial cell line-derived neurotrophic factor

The purpose of this study was to develop a biodegradable polymer nerve guide that locally delivers bioactive neurotrophic factors in physiologically relevant concentrations for the period required by transected peripheral nerves to cross from the proximal to distal nerve stump. Delivery of a neurotrophic factor may enhance nerve regeneration and could potentially be used to overcome the current limitations in nerve repair across large defects. Glial Cell Line-Derived Neurotrophic Factor (GDNF) is a known promoter of axonal elongation and branching and has shown promising pre-clinical results in analysis of nerve regeneration with nerve guides. In addition, GDNF has been shown to promote Schwann cell proliferation and migration. In this study we have created a double-walled microsphere delivery system for bioactive GDNF with a sustained release profile>50 days in vitro. Microspheres were incorporated within degradable poly(caprolactone) nerve guides in a reproducible distribution. Implantation of nerve guides across a 1.5 cm defect in a rat sciatic nerve gap resulted in an increase in tissue integration in both the proximal and distal segments of the lumen of the nerve guide after 6 weeks. In addition, transverse sections of the distal region of the explanted guides showed the presence of Schwann cells while none were detectable in negative control guides. Migration of Schwann cells to double-walled microspheres indicated that bioactive GDNF was encapsulated and delivered to the internal environment of the nerve guide. Because GDNF increased tissue formation within the nerve guide lumen and also promoted the migration and proliferation of Schwann cells, the nerve guides presented within this study show promise toward the development of an off-the-shelf product alternative that promotes nerve regeneration beyond that capable with currently available nerve guides.

Diffusion of soluble factors through degradable polymer nerve guides: Controlling manufacturing parameters.

Nerve guides are cylindrical conduits of either biologically based or synthetic materials that are used to bridge nerve defects. While it is well known that a critical aspect of nerve regeneration is the delivery of oxygen and nutrients to the surviving nerve tissue, several guide parameters that determine the permeability of nerve guides to nutrients are often overlooked. We have reproducibly manufactured poly(caprolactone) (PCL) nerve guides of tailored porosity percentage, wall thickness and pore diameter through a dip-coating/salt-leaching technique. In this study, these three parameters were varied to measure the response of glucose and lysozyme diffusion through the guide wall. In addition, nerve guide permeability following protein fouling studies was examined. Based on the results from this study, it was determined that at high porosity percentages (80%), decreasing the pore diameter (10-38microm) was a measurable method of decreasing the lysozyme permeability of PCL nerve guides while not creating a loss of glucose permeability. PCL fouling studies were used to fine-tune the desirable nerve guide wall thickness. Results indicated that nerve guides 0.6mm thick decreased the loss of lysozyme to almost 10% without significantly diminishing glucose (nutrient) permeability. These results will be utilized to optimize nerve guide parameters for future in vivo applications.

Silk Fibroin Conduits: A Cellular and Functional Assessment of Peripheral Nerve Repair

Silk fibroin conduits were designed with appropriate porosity for peripheral nerve repair. The aim of this work was to use these conduits to examine cell inflammatory responses and functional recovery in a sciatic nerve defect model. A total of 45 randomized Lewis rats were used to create an 8-mm defect bridged by a silk guide, commercial collagen guide, or an autograft. After 1, 4, and 8 weeks, macrophage recruitment, percentage of newly formed collagen, number of myelinated axons, and gastrocnemius muscle mass were evaluated. Following 8 weeks, ED1+ cells in autograft and silk conduits decreased to <1% and 17% of week 1 values, respectively. Collagen formation revealed no difference for all measured time points, suggesting a similar foreign body response. Myelinated axon counts within the silk guide revealed a greater number of proximal spouts and distal connections than collagen guides. Gastrocnemius weights demonstrated a 27% decrease between silk and autografts after 8 weeks. This study demonstrates that, in addition to tailorable degradation rates, our silk conduits possess a favorable immunogenicity and remyelination capacity for nerve repair.

Protein bioactivity and polymer orientation is affected by stabilizer incorporation for double-walled microspheres

Double-walled microspheres present an improved drug delivery technique for sustained release of encapsulated substrates. In this study, the release kinetics and biological activity of lysozyme was analyzed from microspheres comprised of poly(lactic-co-glycolic acid) (PLGA) and poly(L-lactide) (PLLA). In addition, coencapsulation of the anionic surfactant, docusate sodium salt (AOT), was investigated as a method of decreasing protein denaturation during microsphere fabrication. Herein, we show that through the inclusion of AOT, the capacity for two chemically similar polymers to phase separate and form double-walled (DW) microspheres is impaired leading to unique protein release kinetics. Additionally, we present the time period over which our released enzyme, lysozyme, remains biologically active. The consequences of AOT on protein bioactivity are also assessed and provide strong implications for the importance of appropriate stabilizer analysis in future studies involving drug co-encapsulates in polymer based microsphere systems.

A review of adipocyte lineage cells and dermal papilla cells in hair follicle regeneration.

Alopecia is an exceedingly prevalent problem effecting men and women of all ages. The standard of care for alopecia involves either transplanting existing hair follicles to bald areas or attempting to stimulate existing follicles with topical and/or oral medication. Yet, these treatment options are fraught with problems of cost, side effects, and, most importantly, inadequate long-term hair coverage. Innovative cell-based therapies have focused on the dermal papilla cell as a way to grow new hair in previously bald areas. However, despite this attention, many obstacles exist, including retention of dermal papilla inducing ability and maintenance of dermal papilla productivity after several passages of culture. The use of adipocyte lineage cells, including adipose-derived stem cells, has shown promise as a cell-based solution to regulate hair regeneration and may help in maintaining or increasing dermal papilla cells inducing hair ability. In this review, we highlight recent advances in the understanding of the cellular contribution and regulation of dermal papilla cells and summarize adipocyte lineage cells in hair regeneration.

The role of adipose-derived stem cells in endometrial cancer proliferation

Abstract Background. Obesity has been identified as a key risk factor for the development of endometrial cancer (EC), the most common gynecologic malignancy in the US. We hypothesized that adipose tissue from EC patients secretes higher levels of cancer-promoting factors than healthy adipose tissue and promotes tumor cell growth. Methods. In this study, we generated conditioned media from adipose-derived stem cells (ASCs), an important regenerative cell population within adipose tissue. ASCs were isolated from adipose tissue from two EC patients undergoing hysterectomies and four cancer free control patients undergoing elective abdominoplasties. Ishikawa cells were then cultured for 48 hours in ASC-conditioned media (ASC-CM). Study outcomes included cancer cell proliferation rates and angiogenic factor secretion from cancer cells. Results. Our results indicate that ASC-conditioned media significantly increased Ishikawa cell proliferation rate when compared to control Ishikawa culture conditions (p = 0.002). Though not significant, Ishikawa proliferation with conditioned media from EC ASCs was higher than proliferation in conditioned media from control ASCs. Additionally, we found that Ishikawa cells secreted almost 10 % more vascular endothelial growth factor (VEGF) when cultured in EC ASC-CM as compared to Ishikawa cells cultured in healthy (cancer free control) ASC-CM. These results indicate that ASC paracrine factors may positively increase cancer cell growth rate and potentially enhance tumor angiogenesis. Conclusions. Our findings support the hypothesis that adipose tissue is an important source of secreted factors, which increase the rate of EC cell growth. This study provided preliminary evidence that ASCs may be an important parameter to evaluate in relation to EC development.

Adipose stem cell function maintained with age: An intra-subject study of long-term cryopreserved cells

Background The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age. Objectives We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years. Methods SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated. Results The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation. Conclusions By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved. Level of Evidence 5.

Three-Dimensional Adipocyte Culture: The Next Frontier for Adipocyte Biology Discovery.

It is now abundantly clear that adipocytes have extraordinary endocrine and metabolic functions that extend far beyond a simple role in energy storage. Indeed, adipocytes receive and respond to a variety of local and systemic metabolic, hormonal, and neuronal cues. To add further complexity, adipocytes not only exhibit a broad range of cellular diversity along the spectrum of white to beige/brite to brown (1) but also possess unique functional, secretory, and gene expression signatures that are highly dependent on their depot of origin. Numerous differences between adipose tissue depots, notably subcutaneous (sc) and visceral adipose tissues, have been documented (2). However, the mechanisms by which depotspecific differences influence adipocyte biology, and vice versa, remain poorly understood. Traditionally, studies designed to characterize adipocyte biology in isolation from other native tissue structures have relied on culturing preadipocytes under agonistic two-dimensional (2D) culture conditions involving adherence of adipocytes to tissue culture-treated polystyrene plates. Whereas such 2D approaches have been highly successful in elucidating the biology of sc adipocytes, these approaches have been suboptimal for recapitulating the biology of adipocytes from less robust sources such as visceral adipose tissue. This problem results largely because stem cells or preadipocytes isolated from visceral adipose tissue have impaired function with regard to both proliferation and differentiation in standard 2D culture (3, 4). The absence of robust methods to study visceral adipocytes in culture has impeded progress in understanding the full spectrum of biological differences among adipocytes from functionally different yet physiologicallyrelevant adipose tissue depots. Therefore, alternative methods are needed for culturing and studying adipocytes with intrinsic differences due to innate characteristics of the depot of origin. In this issue of Endocrinology, Emont et al (5) describe a facile method for culturing and studying visceral adipocytes in an optimized three-dimensional (3D) collagen hydrogel. Although various forms of 3D hydrogel systems for adipocyte culture have been studied (6, 7), the ability to culture and differentiate visceral adipocytes has remained challenging. Emont et al hypothesize that because visceral preadipocytes differentiate into large and unilocular lipid droplet-containing adipocytes, more 3D extracellular support is required than for sc preadipocytes, which are able to expand and differentiate on rigid polystyrene plates. Their data show that a 3D collagen hydrogel culture system may indeed be superior to a 2D culture system for adipocyte differentiation and culture. Specifically, expression of differentiation and metabolic markers improved substantially in 3D-cultured visceral adipocytes to levels comparable with sc adipocytes. Furthermore, the expression of several markers increased in 3D-cultured sc adipocytes as well. Perhaps more importantly, 3D-cultured adipocytes retained characteristics that more closely resembled their depot of origin. For example, sc adipocytes had higher expression of brown adipocyte-selective markers, and visceral adipocytes had higher expression of inflammatory markers. Finally, assessment of adrenergic-stimulated lipolysis confirmed that these 3Dcultured adipocytes not only maintain morphological characteristics and gene expression profiles of their depot of origin but also important functional characteristics.