Browsing by Subject "Stem cells"

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Hutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary causes of death are stroke and cardiovascular disease at an average age of 14 years. It is known that loss or malfunction of smooth muscle cells (SMCs) in the vasculature leads to cardiovascular defects, however, the exact mechanisms are still not understood. The contribution of other vascular cell types, such as endothelial cells, is still not known due to the current limitations of studying such a rare disorder. Due to limitations of 2D cell culture, mouse models, and the limited HGPS patient pool, there is a need to develop improved models of HGPS to better understand the development of the disease and discover novel therapeutics.

To address these limitations, we produced a functional, three-dimensional tissue model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived cell sources from HGPS patients. To isolate the specific effects of HGPS SMCs, we initially used human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor and iPSC-derived SMCs (iSMCs). TEBVs fabricated from HGPS patient iSMCs and hCB-EPCs (HGPS iSMC TEBVs) showed disease attributes such as reduced vasoactivity, increased medial wall thickness, increased calcification, excessive extracellular matrix protein deposition, and cell apoptosis relative to TEBVs fabricated from primary mesenchymal stem cells (MSCs) and hCB-EPCs or normal patient iSMCs with hCB-EPCs. Treatment of HGPS iSMC TEBVs for one week with the rapamycin analog Everolimus (RAD001), increased HGPS iSMC TEBV vasoactivity and iSMC differentiation in TEBVs.

To improve the sensitivity of our HGPS TEBV model and study the effects of endothelial cells on the HGPS cardiovascular phenotype, we adopted a modified differentiation protocol to produce iPSC-derived vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) from normal and Progeria patient iPSC lines to create iPSC-derived vascular TEBVs (viTEBVs). Normal viSMCs and viECs showed structural and functional characteristics of vascular SMCs and ECs in 2D culture, while HGPS viSMCs and viECs showed various disease characteristics and reduced function compared to healthy controls. Normal viTEBVs had comparable structure and vasoactivity to MSC TEBVs, while HGPS viTEBVs showed reduced vasoactivity, increased vessel wall thickness, calcification, apoptosis and excess ECM deposition. In addition, HGPS viTEBVs showed markers of cardiovascular disease associated with the endothelium such as decreased response to acetylcholine, increased inflammation, and altered expression of flow-associated genes.

The treatment of viTEBVs with multiple Progeria therapeutics was evaluated to determine the potential of the HGPS viTEBV model to serve as a platform for drug efficacy and toxicity testing as well as to further elucidate the mechanisms behind each drugs mode of action. Treatment of viTEBVs with therapeutic levels of the farnesyl-transferase inhibitor (FTI), Lonafarnib, or Everolimus improved different aspects of HGPS viTEBV structure and function. Treatment with Everolimus alone increased response to phenylephrine, improved SMC differentiation and cleared progerin through autophagy. Lonafarnib improved acetylcholine response, decreased ECM deposition, decreased calcification and improved nitric oxide production. Most significantly, combined therapeutic treatment with both drugs showed an additive effect by improving overall vasoactivity, increasing cell density, increasing viSMC and viEC differentiation, and decreasing calcification and apoptosis in treated HGPS viTEBVs. On the other hand, toxic doses of both drugs combined resulted in significantly diminished HGPS viTEBV function through increased cell death. In summary, this work shows the ability of a tissue engineered vascular model to serve as an in vitro personalized medicine platform to study HGPS and potentially other rare diseases of the vasculature using iPSC-derived cell sources. It has also further identified a potential role of the endothelium in HGPS. Finally, this HGPS viTEBV model has proven effective as a drug testing platform to determine therapeutic and toxic doses of proposed therapeutics based on their specific therapeutic effects on HGPS viTEBV structure and function.

The intervertebral discs (IVD) contribute to structural stability of the spinal column, attenuate the impact of compressive loads, and enable a wide spectrum of motions. As a consequence of aging, the majority of the adult population experiences painful symptoms associated with IVD degeneration - a condition characterized by diminished integrity of tissue components. Current treatment options unfortunately cannot restore IVD structure and function. At the present, an avenue of great interest involves autologous or allogeneic cell delivery to the degenerated IVD. Induced pluripotent stem cells (iPSCs) have demonstrated their capacity to differentiate into various cell types. A posited strategy for regenerative medicine applications entails deriving iPSCs from a patient's own somatic cells and directing them toward a specific lineage.

The overall objective of this study is to assess the potential of mouse iPSCs to regenerate nucleus pulposus (NP) cells of the IVD. Previous work identified CD24 as an NP marker, while recent data from our lab noted its expression in mouse iPSCs. The first portion of this thesis employed magnetic activated cell sorting (MACS) to isolate a CD24high iPSC population. Notochordal gene expression was analyzed in this undifferentiated cell fraction via real time RT-PCR. Mouse iPSCs were then cultured in a laminin-rich, 3D culture system for up to 28 days, and NP phenotype was assessed by immunostaining.

The latter half of this work focused on producing a more conducive environment for NP differentiation of mouse iPSCs. This involved the addition of low oxygen tension and notochordal conditioned medium (NCCM) to the culture platform. Mouse iPSCs were evaluated for ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Furthermore, they were compared to NIH 3T3 mouse embryonic fibroblasts cultured under the same conditions.

Results demonstrated that a CD24high fraction of mouse iPSCs could be successfully retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of hypoxia and NCCM generated similar phenotypic results. 3T3 fibroblasts unexpectedly exhibited transdifferentiation potential as well. Altogether, these studies conclude that mouse iPSCs do have potential to differentiate into NP-like cells and may be applied to future cell-based therapies for restoration of the degenerated IVD.

Articular cartilage is the thin, load-bearing connective tissue that lines the ends of long bones in diarthroidal joints, providing predominantly a mechanical function. Because cartilage is avascular and aneural, it has little capacity for self-repair if damaged. One repair strategy is through a functional tissue engineering approach using adipose-derived stem cells (ASCs). ASCs are an abundant progenitor cell source easily obtained through a minimally invasive liposuction procedure. When appropriately stimulated, ASCs have demonstrated significant potential for chondrogenic differentiation. Though studies have demonstrated the ability of ASCs to synthesize cartilage-specific macromolecules, a more thorough understanding of factors that modulate ASC chondrogenesis is required. Accordingly, the central aim of this dissertation was to study the chondrogenic response of ASCs to biochemical, biomechanical, and biomaterial factors.

We hypothesized that factors, other than TGF-beta and dexamethasone, would improve ASC chondrogenesis. BMP-6 emerged as a potent regulator of ASC chondrogenesis, particularly in early culture, as noted by significant upregulation of cartilage-specific extracellular matrix (ECM) genes and downregulation of cartilage hypertrophy markers.

Hypothesizing that biomechanical factors would accelerate the formation of cartilage-specific macromolecules, we designed and manufactured an instrument to apply dynamic deformational loading to ASC seeded constructs. Dynamic loading significantly inhibited ASC metabolism and downregulated cartilage-specific ECM genes. However, 21 days of dynamic loading induced the production of type II collagen, a principal component of articular cartilage.

We hypothesized that a biomaterial derived from cartilage would serve as a bioactive scaffold and induce chondrogenic differentiation. The novel, ECM-derived scaffold promoted the most robust differentiation of ASCs relative to both biochemical and biomechanical factors, particularly noted by a type II collagen-rich matrix after 28 days of culture. After 42 days of culture, biphasic mechanical testing revealed an aggregate modulus of 150 kPa, approaching that of native cartilage. These data suggest that the ECM-derived scaffold may retain important signaling molecules to drive differentiation or that ASC differentiation is dependent on proper cell anchorage.

In summary, we have shown that biochemical, biomechanical, and biomaterial factors have strong influences on the chondrogenic potential of ASCs. Optimization of these factors will ultimately be required to successfully engineer a functional tissue.

The lungs are vital organs whose airways are lined with a continuous layer of epithelial cells. Epithelial cells in the distal most part of the lung, the alveolar space, are specialized to facilitate gas exchange. Proximal to the alveoli is the airway epithelium, which provides an essential barrier and is the first line of defense against inhaled toxicants, pollutants, and pathogens. Although the postnatal lung is a quiescent organ, it has an inherent ability to regenerate in response to injury. Proper balance between maintaining quiescence and undergoing repair is crucial, with imbalances in these processes leading to fibrosis or tumor development. Stem and progenitor cells are central to maintaining balance, given that they proliferate and renew both themselves and the various differentiated cells of the lung. However, the precise mechanisms regulating quiescence and repair in the lungs are largely unknown. In this dissertation, ionizing radiation is used as a physiologically relevant injury model to better understand the repair process of the airway epithelium. We use in vitro and in vivo mouse models to study the response of a secretory progenitor, the club cell, to various doses and qualities of ionizing radiation. Exposure to radiation found in space environments and in some types of radiotherapy caused clonal expansion of club cells specifically in the most distal branches of the airway epithelium, indicating that the progenitors residing in the terminal bronchioles are radiosensitive. This clonal expansion is due to an increase in p53-dependent apoptosis, senescence, and mitotic defects. Through the course of this work, we discovered that p53 is not only involved in radiation response, but is also a novel regulator of airway epithelial homeostasis. p53 acts in a gene dose-dependent manner to regulate the composition of airway epithelium by maintaining quiescence and regulating differentiation of club progenitor cells in the steady-state lung. The work presented in this dissertation represents an advance in our understanding of the molecular mechanisms underlying maintenance of airway epithelial progenitor cells as well as their repair following ionizing radiation exposure.

Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Identifying new treatments will require an understanding of early stages of tumor development - the cell types from which the tumors arise and the signals that regulate their growth - as well as identification of pathways that are critical for the growth and maintenance of established tumors.

In these studies, we first explore the role of WNT signaling in cerebellar progenitors and their potential to serve as cells of origin for WNT-driven tumors. The WNT pathway plays multiple roles in neural development, is crucial for establishment of the embryonic cerebellum, and is highly expressed in a subset of MBs. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. We show that expression of activated β-catenin promotes proliferation of cerebellar neural stem cells (NSCs) but not granule neuron precursors (GNPs). Although β-catenin expressing NSCs proliferate in vivo they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation and that cooperating "second hits" may be required for neoplastic transformation.

In addition to understanding early stages of transformation, identifying vulnerabilities of established tumors will be critical for development of targeted therapies. Our studies in Chapter 3 are focused on the role of Survivin in SHH-driven MB and utility of survivin inhibition as a therapeutic approach for MB. Survivin is an inhibitor of apoptosis protein (IAP) that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB, and when expressed at high levels predicts poor clinical outcome. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Using genetic and pharmacological approaches, we demonstrate that inhibition of Survivin impairs proliferation and survival of both murine and human MB cells. Although Survivin antagonists do not cross the blood-brain barrier, they are capable of impeding growth of MB cells in flank allografts. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.

Vertebrates like urodele and teleost have an enhanced capacity for regeneration, when compared to mammals. Recently, the teleost zebrafish (Danio rerio) has become a popular model for studying regenerative events, due to the ability to regenerate multiple organs such as the fin and the heart, and the diverse genetic approaches available for functional studies. In my thesis studies, I have used the zebrafish caudal fin as a model system to understand molecular and cellular mechanism of appendage regeneration.

Pharmacological and genetic studies have revealed that Fgf signaling is important for appendage regeneration. To dissect the mechanism of Fgfs during zebrafish fin regeneration, lab colleagues and I have generated and utilized transgenic animals in which Fgf signaling can be experimentally increased or decreased. Through these transgenic studies, I found that position-dependent Fgf signaling directs regenerative growth and blastemal proliferation. Proximally-amputated fin regenerates grow at higher rates than the distally-amputated, owing to position-dependent amounts of Fgf activity. Further studies using new transgenics have provided an understanding of mechanisms by which Fgfs influence epidermal regulation of the blastema. Loss- and gain-of-function studies of Fgfs reveal that Fgf signaling both positively and negatively regulated shh expression in the epidermis to maintain blastemal function.

During the fin regeneration process, pigmentation pattern is re-established as along with bone structures and connective tissues. While the lineage of the blastema is not precisely clear, pigment cells in the fin regenerates are thought to be derived from melanocyte stem cells. Therefore, melanocyte regeneration is an informative system to understand the mechanism underlying regulation of adult stem cells during regeneration. As part of my thesis studies, we generated transgenic animals in which ectopic Ras expression can be experimentally induced. Transgenic studies, combined with pharmacological approaches, have revealed that Ras controls self-renewal of melanocyte stem cells during fin pigment regeneration.

Obesity due to a high-fat diet is characterized by accumulation of inflammatory macrophages in tissues, leading to chronic low-grade systemic inflammation. Obese individuals also exhibit impaired tissue healing. With a high-fat feeding, cells are exposed to the elevated levels of dietary fatty acids (FAs), and such a change of microenvironment may alter their properties. Stem cells are cells capable of multipotent differentiation, and this potential allows them to play a promising role in healing and regenerative medicine. However, the effect of obesity, particularly various types of dietary FAs, on the function of stem cells remains largely unknown. Furthermore, obesity is a primary risk factor of osteoarthritis (OA), a disease of entire of joint involving degradation of cartilage, synovitis, and subchondral bone changes. Yet, the mechanisms linking obesity and OA are not fully understood. Furthermore, although macrophages are well recognized for their inflammatory role in obesity, little is known regarding functionality of these cells in regulating the effect of obesity on OA. This dissertation develops fundamental stem cell isolation and culture techniques, and utilizes animal models to investigate (1) the influences of high-fat diet induced-obesity on function of adult stem cells, (2) examine the effect of obesity and dietary FAs on OA, and (3) evaluate the role of macrophages in obesity-associated OA by depleting macrophages using a transgenic mouse model.

A variety of adult stem cell populations including bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs), and infrapatellar-derived stem cells (IFP cells) were successfully isolated from lean and obese mice and expanded in vitro. Obese stem cells demonstrated altered multilineage differentiation potential and distinct immunophenotypes as compared to lean stem cells. Furthermore, FA treatment of lean stem cells significantly changed their multipotency but did not completely recapitulate the properties of obese stem cells.

Supplementation of ω-3 polyunsaturated fatty acids (PUFAs) in a high-fat diet was capable to mitigate injury-induced OA and decrease serum inflammatory cytokine levels. ω-3 PUFAs also significantly enhanced wound repair, while saturated FAs and ω-6 PUFAs act as a detrimental factor in OA, synovitis, and wound healing. Spontaneous locomotion of the mice was independent of OA development. Furthermore, using mathematical models and weight-matched mice, we found that OA was significantly associated with dietary FA content but not with body weight and mouse activity. These results suggest that metabolic factor plays a more significant role in obesity-associated OA than mechanical factor.

Despite their temporary improved metabolic parameters and reduced osteophyte formation, obese mice receiving short-term, systemic macrophage depletion did not mitigate cartilage degeneration following joint injury. Instead, macrophage depletion significantly enhanced joint synovitis in the surgery-operated joint. Macrophage-depleted mice also exhibited up-regulated expression of inflammatory cytokines in synovial fluid. These findings indicate that despite their recognized pro-inflammatory role, macrophages are vital in regulating the homeostasis of immune cells in the joint following injury.

Taken together, this research further elucidates the relationships among obesity, stem cells, and OA. The results from our study may provide a framework to develop stem cell therapy for obese patients and intervention program for obese OA patients in the future.