Browsing by Subject "Stem Cells"

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3D Printing to Recapitulate Cardiac Tissue Development, Structure, and Function
(2019-09) Kupfer, Molly
Heart disease is the leading cause of death worldwide, due in large part to the low regenerative capacity of the heart. With recent advances in stem cell biology, cardiac tissue engineering with human cells has emerged as an avenue to replace lost muscle after a cardiac event and to produce in vitro, human models for drug and medical device testing. However, efforts in this realm are still limited in their ability to recapitulate the complex, microscale interactions that enable macroscale function of cardiac muscle. 3D printing is a technology that is poised to meet this challenge, as it enables precise deposition of cues that are critical for cells to connect with each other and engage with their environment. Here we present three studies that capitalize on the replicative power of 3D printing as tool to advance the functionality of engineered cardiac tissues by promoting connections between cardiomyocytes, supporting cells of the heart, and the extracellular matrix. The foundation of this work lies in our view that the generation of physiologically relevant tissue mimics requires a robust mechanistic understanding of how these systems develop in vivo, and how the vital interactions that occur between differentiating cells and their environment can be recapitulated in vitro. Doing so will enable us to address critical gaps in field of cardiac tissue engineering while advancing clinical models and therapeutics.
Dynamic Changes in DNA Replication Timing and 3D Genome Organization During Cardiac Differentiation
(2023-03) Martinez Cifuentes, Santiago
Genome architecture has emerged as a key factor of gene regulation and is tightly coordinated with the temporal order of DNA replication (Replication Timing – RT). RT and 3D genome organization change dynamically throughout development in correlation with the establishment of cell type specific gene expression patterns. The first organ to develop during embryonic development is the heart. The heart is a post-mitotic, terminally differentiated organ which, compared to other organs such as the liver, lacks the capacity to regenerate upon injury such as myocardial infarction. Instead, it forms fibrotic tissue that maintains organ integrity but undermines pump function, often leading to congestive heart failure and premature death. Deciphering the 3D genome organization in cardiac differentiation may help our understanding of gene regulation during cardiac development. DNA replication timing (RT) is a very informative functional readout of large-scale chromatin organization across distinct cell types and its regulation during development. RT is cell type-specific, highly conserved and changes in RT affect approximately half the genome during development and differentiation. I hypothesize that changes in the 3D genome organization play a critical role in gene regulation and cell function in cardiac differentiation. In this work, I will differentiate human embryonic stem cells (hESCs) towards cardiomyocytes and use a multiomics approach (RT, transcriptome and 3D genome organization) to identify the dynamic changes in genome architecture, RT and gene expression during normal cardiac development. This will allow us, in the future, to construct an integrative model of nuclear function in cardiac cells that can be leveraged as a framework to identify cellular alterations associated with congenital cardiovascular diseases.
The effects of wheat class and processing on markets of colon cancer risk in carcinogen-treated rats.
(2009-02) Islam, Ajmila
A previous study in this laboratory found that hard red wheat is more effective than soft white wheat in reducing colon cancer risk, regardless of processing state, based on fewer aberrant crypt foci (ACF), a morphological marker of colon cancer risk. Here we examined the effect of wheat class (red vs. white) and processing (whole vs. refined) on reducing markers of colon cancer risk during the early and late promotion stage of colon cancer development. Rats adapted to a basal diet were treated twice with the colon-specific carcinogen, dimethylhydrazine (DMH). After the last dose of carcinogen, rats were divided into either the basal diet or the wheat flour-based diet groups. Both hard red and soft white wheat flour significantly reduced morphological markers such as ACF, and sialomucin producing ACF (SiM-ACF), an ACF with greater tumorigenic potential, compared to the basal diet. These reductions occurred equally with whole and refined wheat. Both hard red and soft white wheat diets significantly reduced a biochemical marker of risk, beta-catenin accumulated crypts (BCAC), compared to the basal diet, but hard red wheat did so to a greater degree. Only hard red wheat significantly reduced a marker of stem cells mutation, metallothionein positive crypts, compared to soft white wheat. Hard red wheat caused regression of ACF, suggesting it can reduce the risk level of colon cancer. Overall, hard red wheat reduced colon cancer risk more than soft white wheat, regardless of processing state. The differences between wheat flours were greater in the late promotion stage.
Engineering human pluripotent stem cells for enhanced lymphocyte development and function
(2012-10) Knorr, David Arthur
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide accessible, genetically tractable and homogenous starting cell populations to efficiently study human blood cell development. These cell populations provide platforms to develop new cell-based therapies to treat both malignant and non-malignant hematological diseases. Our group has previously demonstrated the ability of hESC-derived hematopoietic precursors to produce functional natural killer (NK) cells. hESCs and iPSCs, which can be reliably engineered in vitro, provide an important model system to study human lymphocyte development and produce enhanced cell-based therapies with potential to serve as a "universal" source of anti-tumor lymphocytes for novel clinical therapies. My studies have focused on the generation of NK cells from hESCs and iPSCs, their function both in vitro and in vivo against a variety of different tumor types, and modification of these cells with genetic constructs to enhance their anti-tumor capabilities.
Human stem cells: an ethical overview
(University of Minnesota, Center for Bioethics, 2003) University of Minnesota: Center for Bioethics
An improved method for generating oligodendrocyte progenitor cells from murine induced pluripotent stem cells
(2014-01) Terzic, Dino
Cell based therapies aiming to restore myelin in the central nervous system offer great hope for treatment of numerous conditions, ranging from multiple sclerosis and the leukodystrophies, to traumatic CNS injury. The oligodendrocyte progenitor cell (OPC) gives rise to functional oligodendrocytes following transplantation into dysmyelinated regions of the central nervous system, and as such represents a candidate for potential therapeutic applications. It can be generated by directed differentiation of a variety of pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells. The iPS cell is an ideal source for derivation of OPCs, as it offers the advantage of autologous transplants, and a model to study the biology and pathology of OPCs and oligodendrocytes. Existing protocols for deriving OPCs from mouse iPS cells, although a valuable model, are inefficient and not easily reproducible. We improved upon the existing differentiation protocols to increase their consistency and yield of OPCs, by modifying and combining several published methods. We demonstrate robustness of our new method by generating OPCs from several different pluripotent stem cell lines and demonstrating that the OPCs further develop to form functional oligodendrocytes in vivo.
Investigating the use of multipotent adult progenitor cells for treatment of Duchenne muscular dystrophy: a translational approach.
(2008-07) Frommer, Sarah Anne
Taking a “translational” approach to developing clinical therapies is a two step process that requires: 1) Basic science research on clinical diseases; and 2) application of knowledge gained or resultant therapeutics from that research to patient care. The collaboration of basic sciences and clinical sciences will result in greater advancement of knowledge within each field. There are many diseases that have no cure, even with the tools that modern medicine has to offer. A good example of this is Duchenne muscular dystrophy (DMD). Unfortunately, there is no cure and no effective long-term treatment to delay the progression of DMD; modern medicine can only ameliorate the symptoms and attempt to give the patient the best quality of life possible. It may one day be possible to cure patients if even one of the many experimental therapies for DMD, aimed at restoring dystrophin in skeletal muscle and shown to improve muscle function in dystrophic animals, could be developed clinically. One such therapy is stem cell therapy. The stem cells used in this work are multipotent adult progenitor cells (MAPC). MAPC were first discovered in the Verfaillie lab here at the University of Minnesota- Twin Cities. It was traditionally believed that adult stem cells like hematopoietic stem cells and mesenchymal stem cells could not differentiate into cells outside the mesodermal lineage; however there are currently numerous reports that challenge this thought. This thesis presents the application of a three-step translational approach toward development of stem cell therapy for treatment of DMD. The three steps are: 1) In vitro study of MAPC myogenic potential; 2) in vivo study of MAPC myogenic potential; and 3) development of a system to measure functional effects of therapies. Chapter 2 describes multifactorial testing of different cytokines in an effort to develop a protocol aimed at directing myogenic induction. Chapter 3 describes methods developed and subsequently tested to enhance MAPC engraftment in the DMD model mouse upon intramuscular injection. Chapter 4 describes the development and testing of a system aimed at detecting functional differences due to therapy.
A Systems Approach to Studying the Differentiation of Stem Cells Towards Hepatocytes
(2017-08) Chau, David
The recent advancements in stem cell biology have allowed for new and exciting opportunities to use stem cells in clinical and industrial applications. Stem cells have the unique ability to self-renew and differentiate into any specialized cell type found in the body. Using certain mechanical and biochemical cues, stem cells can be directed to become any specific cell type, such as hepatocytes. A robust and efficient process for expansion and differentiation to generate large quantities of functional hepatocytes from stem cells will be essential to establishing a stem cell bioprocess in the future for therapeutic and industrial applications of hepatocytes. In this study, a differentiation protocol with soluble growth factors and cytokines was used to mimic the key signaling cues during embryonic development. However, most directed differentiation processes have run into issues with limited scalability and lack of functionality in the differentiated cells. In an effort to bring stem cell therapy closer to reality, our strategy was to use a systems-based approach to enhance the quality and yield of stem cell-derived hepatocytes. To achieve higher cell yield, we modified an existing differentiation protocol to incorporate a cell expansion stage to facilitate simultaneous differentiation and cell growth. Using transcriptome analysis and mass cytometry, we showed how the population of cells changed over time on both the transcript and protein level. Both analyses revealed that with the new expansion stage, we obtained a higher quantity of hepatocytes within the same time frame compared to the conventional method of differentiation. We then showed the capability to scale up our differentiation for larger scale cultures by adapting the expansion stage onto Cytodex 3 microcarriers. Using the same culture volume as a tissue plate culture, we demonstrated the ability to achieve up to a 5-fold increase in cell number with a final cell density in the range of 4-5x106 cells/ml. These strategies show that the demand for large quantities of hepatocytes can be met by translating the conventional method of differentiation to suspension microcarrier differentiation. Encouraged by our ability to yield higher cell density using microcarrier culture, we explored assessing the functional maturity of our stem cell-derived hepatocytes using transcriptome analysis. We showed that stem cell-derived hepatocytes are still clearly different when compared to primary hepatocytes at the transcriptome level. In addition to evaluating cells using transcriptome analysis, we wanted to be able to compare the current in-vitro processes to embryonic liver development to understand the genetic roadblocks. The transcriptome data from hESCs hepatocyte differentiation was integrated with mouse liver development using principal component analysis and batch corrections. This allowed us to create a unified developmental scale to compare samples from different species and in-vitro to in-vivo platforms. The meta-analysis revealed that stem cell-derived hepatocytes are equivalent to the functional maturity of developing cells at E15 in mouse development. From the transcriptome analysis, we observed many different genes in energy metabolism with dynamic behavior over the course of differentiation. We sought to understand the effect of changes in different metabolic genes and the impacts on metabolic transition during differentiation. We characterized the energy metabolism of hESCs and assessed the metabolic demand of cells at different stages of differentiation. hESCs and early differentiated cells exhibited a high glycolytic flux. transitioning towards an oxidative metabolism as the differentiation progressed. Furthermore, using confocal microscopy, we also characterized the activity and morphology of the mitochondria in the cells at different stages of differentiation. Using the consumption rates of different nutrients as an input to our metabolic flux model along with our transcriptome findings, we were able to gain a deeper understanding of the metabolic behavior of cells during differentiation. Our analysis revealed that cells consume lower amounts of glucose over the course of the differentiation but become more efficient at transporting pyruvate into the mitochondria leading to increased oxidative phosphorylation. However, our metabolic and transcriptome data revealed that our stem cell-derived hepatocytes are not capable of mature metabolic functions such as gluconeogenesis, supporting the immature phenotype that has been described in literature. Together, these studies reveal that stem cells can provide a renewable and scalable source of hepatocytes for therapeutic applications. These cells demonstrate some phenotypic and functional properties of primary hepatocytes but have some contrasting elements compared to their in-vivo counterparts that will need further genetic intervention to enhance their maturation before cellular therapy can become a reality. However, this work is invaluable as it contributes to the current status of the field and facilitates the translation of laboratory practices of stem cell culture into a scalable technology.
Systems Engineering of Stem Cell Fate
(2015-08) Raju, Ravali
Recent advances in the derivation of functional cells from pluripotent stem cells have raised hope for cell therapy to treat liver ailments. They have enhanced the prospects of developing reliable in vitro models for liver diseases and drug toxicity screening. A differentiation protocol mimicking key signaling cues of embryonic development was developed to direct stem cells (ES) towards the hepatic fate and express key hepatic markers and functions. While these results are encouraging, most directed differentiations from stem cells to the target cell types are hampered by lack of functional maturity, cell heterogeneity and low cell yields limiting their translation to the clinic. These cells are therefore refereed to hepatocyte-like cells (HLCs). An integrative strategy was employed including both experimental techniques as well as a systems-based analysis towards enhancing the product quality and yields of HLCs. Functional maturity was enhanced by initiating three dimensional spheroid formation upon differentiation. Enrichment of hepatic cells using selective medium conditions was performed to obtain higher fraction of cells with the desired properties. Cell expansion was incorporated during differentiation to improve cell yields. Several additional strategies have been used to increase hepatocyte maturity in literature including co-culture and transfection with transcription factors. These methods including ours have shown improvement, however a universal gap to maturation is still present when compared to primary hepatocytes. Comparison of transcriptome data of differentiation to embryonic liver development can elucidate the genetic roadblocks preventing ES cells from reaching the functional maturity of their tissue counterparts. Transcriptome data was compiled from various depositories for mouse fetal liver development (from E8.5 to post-natal). Transcriptome data was obtained during the time course of our human hepatic differentiation protocol and was augmented with human in vitro hepatic differentiation data in the public depository. Interestingly, majority of the HLCs are similar irrespective of the cell source and protocol. The entire cohort of HLCs clustered separately from the primary hepatocytes and adult liver indicating an inherent roadblock to maturation. The transcriptome data of human ES hepatic differentiations was then integrated with mouse liver development using a unique approach. This allowed us to identify the corresponding development stage at which the in vitro stem cell differentiation is blocked. The analysis uncovered a pivotal gene set with contrasting profiles in ES differentiation and mouse liver development that merit combinatorial genetic intervention to enhance maturation of ES derived hepatocytes. Thus, one can envision the availability of stem cell based liver therapies in the not so distant future.
Tracking hematopoietic development from human pluripotent stem cells
(2013-11) Ferrell, Patrick Ian
Hematopoietic stem cells (HSCs) are a powerful resource for both regenerative medicine and the study of human developmental biology. Though much is known about HSC physiology and development in mice, experimental limitations make their characterization a greater challenge in human. As such, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are currently the best systems with which to model early human hematopoietic development in vitro, thus providing insight regarding crucial factors delineating HSC emergence, maintenance and subsequent differentiation. However, generation of HSCs from hESCs and iPSCs relies on an intimate understanding of both the in vivo hematopoietic microenvironment as well as HSC phenotype for their prospective isolation. Though current in vitro protocols can readily generate hESC and iPSC-derived cells with hematopoietic progenitor function, none of these populations has exhibited what should be the hallmark of an HSC: robust, long-term, multilineage reconstitution of an immunodeficient recipient upon transplantation. These studies address this issue by using transgenic hESC and iPSC lines which report the expression of genes known to be crucial for early hematopoietic events in mice so that they may help us to understand how they translate to human development in vitro. Furthermore, this effort is complemented by additional studies using hESC-derived stromal populations to provide assays that help assess putative HSC quality, maintenance and the hematopoietic niche.

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