Microfluidic production of stem-cell microcapsules for spinal cord injury repair

Stem cell therapy demonstrates much promise for the replacement of damaged tissue in several diseases, including spinal cord injury. However, challenges around the control of stem cell fate in situ still hinders effective recovery of the normal tissue function. Stem cell encapsulation permits their immobilization within biocompatible scaffolds, allowing for a better control of parameters such as proliferation, integration, migration and differentiation within the host tissue. A customized microfluidic device was developed for the production of alginate microcapsules. The diameter of such microcapsules could be easily controlled by the modification of the fluids flow rates, allowing for the reproducible production of highly monodisperse microcapsules. This microfluidic method was then successfully applied for the encapsulation of two different types of stem cells: (i) Neural Stem Cells and (ii) Dental Pulp Stem Cells. Both cell types demonstrated survival within the alginate microcapsules for up to three weeks in culture. However, an early egress of cells from inside to outside of the microcapsules was observed 3 days post-encapsulation. In order to delay such cell escape, alginate microcapsules were modified through the addition of type I collagen. The alginate-collagen microcapsules permitted similar rates of cell survival and permitted the delay of cell egress until 10 days after encapsulation. Stem cells demonstrated a retention of their stem cell and neuronal differentiation properties upon selective release from alginate-collagen microcapsules, as demonstrated by high proliferation rates and the production of stem cell and neuronal markers. When cell-laden microcapsules were transplanted into an ex vivo SCI model the microcapsules were able to effectively retain the transplanted stem cells at the site of implantation. Transplanted cells survived up to 10 days in culture after transplantation and demonstrated the production of neuronal markers within the cord cultures. The results presented in this thesis demonstrate the ability of stem cells to retain their viability and neuronal differentiation capacity within alginate-collagen microcapsules, thereby providing a promising future therapy for the treatment of spinal cord injury

Microfluidic encapsulation supports stem cell viability, proliferation and neuronal differentiation

Stem cell encapsulation technology demonstrates much promise for the replacement of damaged tissue in several diseases, including spinal cord injury (SCI). The use of biocompatible microcapsules permits the control of stem cell fate in situ to facilitate the replacement of damaged/lost tissue. In this work, a novel customized microfluidic device was developed for the reproducible encapsulation of neural stem cells (NSCs) and dental pulp stem cells (DPSCs) within monodisperse, alginate-collagen microcapsules. Both cell types survived within the microcapsules for up to 21 days in culture. Stem cells demonstrated retention of their multipotency and neuronal differentiation properties upon selective release from the microcapsules, as demonstrated by high proliferation rates and the production of stem cell and neuronal lineage markers. When cell-laden microcapsules were transplanted into an organotypic SCI model, the microcapsules effectively retained the transplanted stem cells at the site of implantation. Implanted cells survived over a 10 day period in culture after transplantation and demonstrated commitment to a neural lineage. Our device provides a quick, effective, and aseptic method for the encapsulation of two different stem cell types (DPSCs and NSCs) within alginate-collagen microcapsules. Since stem cells were able to retain their viability and neural differentiation capacity within such microcapsules, this method provides a useful technique to study stem cell behavior within three-dimensional environments. View Article Tools Share Abstract Stem cell encapsulation technology demonstrates much promise for the replacement of damaged tissue in several diseases, including spinal cord injury (SCI). The use of biocompatible microcapsules permits the control of stem cell fate in situ to facilitate the replacement of damaged/lost tissue. In this work, a novel customized microfluidic device was developed for the reproducible encapsulation of neural stem cells (NSCs) and dental pulp stem cells (DPSCs) within monodisperse, alginate-collagen microcapsules. Both cell types survived within the microcapsules for up to 21 days in culture. Stem cells demonstrated retention of their multipotency and neuronal differentiation properties upon selective release from the microcapsules, as demonstrated by high proliferation rates and the production of stem cell and neuronal lineage markers. When cell-laden microcapsules were transplanted into an organotypic SCI model, the microcapsules effectively retained the transplanted stem cells at the site of implantation. Implanted cells survived over a 10 day period in culture after transplantation and demonstrated commitment to a neural lineage. Our device provides a quick, effective, and aseptic method for the encapsulation of two different stem cell types (DPSCs and NSCs) within alginate-collagen microcapsules. Since stem cells were able to retain their viability and neural differentiation capacity within such microcapsules, this method provides a useful technique to study stem cell behavior within three-dimensional environments

Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal.

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Cell-type-specific quantification of protein synthesis in vivo

Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques to investigating protein synthesis within mammalian systems in vivo has been challenging. The synthesis of O-propargyl-puromycin (OP-Puro), an analog of puromycin that contains a terminal alkyne group, has facilitated the quantification of protein synthesis within individual cells in vivo. OP-Puro enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Incorporated OP-Puro can be detected through a click-chemistry reaction that links it to a fluorescently tagged azide molecule. In this protocol, we describe how to administer OP-Puro to mice, obtain cells of interest (here, we use bone marrow cells) just 1 h later, and quantify the amount of protein synthesized per hour by flow cytometry on the basis of OP-Puro incorporation. We have used this approach to show that hematopoietic stem cells (HSCs) exhibit an unusually low rate of protein synthesis relative to other hematopoietic cells, and it can be easily adapted to quantify cell-type-specific rates of protein synthesis across diverse mammalian tissues in vivo. Measurement of protein synthesis within bone marrow cells in a cohort of six mice can be achieved in 8-10 h