Biofabrication of Artificial Stem Cell Niches in the Anterior Ocular Segment

The anterior segment of the eye is a complex set of structures that collectively act to maintain the integrity of the globe and direct light towards the posteriorly located retina. The eye is exposed to numerous physical and environmental insults such as infection, UV radiation, physical or chemical injuries. Loss of transparency to the cornea or lens (cataract) and dysfunctional regulation of intra ocular pressure (glaucoma) are leading causes of worldwide blindness. Whilst traditional therapeutic approaches can improve vision, their effect often fails to control the multiple pathological events that lead to long-term vision loss. Regenerative medicine approaches in the eye have already had success with ocular stem cell therapy and ex vivo production of cornea and conjunctival tissue for transplant recovering patients’ vision. However, advancements are required to increase the efficacy of these as well as develop other ocular cell therapies. One of the most important challenges that determines the success of regenerative approaches is the preservation of the stem cell properties during expansion culture in vitro. To achieve this, the environment must provide the physical, chemical and biological factors that ensure the maintenance of their undifferentiated state, as well as their proliferative capacity. This is likely to be accomplished by replicating the natural stem cell niche in vitro. Due to the complex nature of the cell microenvironment, the creation of such artificial niches requires the use of bioengineering techniques which can replicate the physico-chemical properties and the dynamic cell–extracellular matrix interactions that maintain the stem cell phenotype. This review discusses the progress made in the replication of stem cell niches from the anterior ocular segment by using bioengineering approaches and their therapeutic implications.

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SALL4 is a robust stimulator for the expansion of hematopoietic stem cells

Blood ◽

10.1182/blood-2011-01-333641 ◽

2011 ◽

Vol 118 (3) ◽

pp. 576-585 ◽

Cited By ~ 55

Author(s):

Jerell R. Aguila ◽

Wenbin Liao ◽

Jianchang Yang ◽

Cecilia Avila ◽

Nabil Hagag ◽

...

Keyword(s):

Stem Cell ◽

Ex Vivo ◽

Constitutive Expression ◽

Granulocytic Differentiation ◽

Hematopoietic Stem ◽

Cell Therapies ◽

Human Hscs ◽

Rare Cells

Abstract HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by >10 000-fold for both CD34+/CD38− and CD34+/CD38+ cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34+ cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.

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Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽

10.3390/cells10040730 ◽

2021 ◽

Vol 10 (4) ◽

pp. 730

Author(s):

Biji Mathew ◽

Leianne A. Torres ◽

Lorea Gamboa Gamboa Acha ◽

Sophie Tran ◽

Alice Liu ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Extracellular Vesicles ◽

Time Course ◽

Ganglion Cells ◽

Ex Vivo ◽

Dependent Manner ◽

Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

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The Therapeutic Implications of Plasticity of the Cancer Stem Cell Phenotype

PLoS ONE ◽

10.1371/journal.pone.0014366 ◽

2010 ◽

Vol 5 (12) ◽

pp. e14366 ◽

Cited By ~ 44

Author(s):

Kevin Leder ◽

Eric C. Holland ◽

Franziska Michor

Keyword(s):

Stem Cell ◽

Cancer Stem Cell ◽

Cell Phenotype ◽

Therapeutic Implications ◽

Cancer Stem Cell Phenotype ◽

Stem Cell Phenotype

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Using ex vivo culture to assess dynamic phenotype changes in human prostate macrophages following exposure to therapeutic drugs

Scientific Reports ◽

10.1038/s41598-021-98903-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Clovis Boibessot ◽

France-Hélène Joncas ◽

Aerin Park ◽

Zohra Berrehail ◽

Jean-François Pelletier ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Cell ◽

Ex Vivo ◽

Cell Phenotype ◽

Gleason Grade ◽

Tumor Resistance ◽

Grade Group ◽

In Vivo Models ◽

Novel Associations

AbstractWithin the prostate tumor microenvironment (TME) there are complex multi-faceted and dynamic communication occurring between cancer cells and immune cells. Macrophages are key cells which infiltrate and surround tumor cells and are recognized to significantly contribute to tumor resistance and metastases. Our understanding of their function in the TME is commonly based on in vitro and in vivo models, with limited research to confirm these model observations in human prostates. Macrophage infiltration was evaluated within the TME of human prostates after 72 h culture of fresh biopsies samples in the presence of control or enzalutamide. In addition to immunohistochemistry, an optimized protocol for multi-parametric evaluation of cellular surface markers was developed using flow cytometry. Flow cytometry parameters were compared to clinicopathological features. Immunohistochemistry staining for 19 patients with paired samples suggested enzalutamide increased the expression of CD163 relative to CD68 staining. Techniques to validate these results using flow cytometry of dissociated biopsies after 72 h of culture are described. In a second cohort of patients with Gleason grade group ≥ 3 prostate cancer, global macrophage expression of CD163 was unchanged with enzalutamide treatment. However, exploratory analyses of our results using multi-parametric flow cytometry for multiple immunosuppressive macrophage markers suggest subgroup changes as well as novel associations between circulating biomarkers like the neutrophil to lymphocyte ratio (NLR) and immune cell phenotype composition in the prostate TME. Further, we observed an association between B7–H3 expressing tumor-associated macrophages and the presence of intraductal carcinoma. The use of flow cytometry to evaluate ex vivo cultured prostate biopsies fills an important gap in our ability to understand the immune cell composition of the prostate TME. Our results highlight novel associations for further investigation.

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Bioengineering Approaches to Accelerate Clinical Translation of Stem Cell Therapies Treating Osteochondral Diseases

Stem Cells International ◽

10.1155/2020/8874742 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Meng Wang ◽

Yixuan Luo ◽

Yin Yu ◽

Fei Chen

Keyword(s):

Mechanical Properties ◽

Stem Cell ◽

Recent Progress ◽

Osteochondral Defect ◽

Cell Phenotype ◽

Stem Cell Therapies ◽

Future Directions ◽

Cell Therapies ◽

Osteochondral Repair ◽

Therapeutic Properties

The osteochondral tissue is an interface between articular cartilage and bone. The diverse composition, mechanical properties, and cell phenotype in these two tissues pose a big challenge for the reconstruction of the defected interface. Due to the availability and inherent regenerative therapeutic properties, stem cells provide tremendous promise to repair osteochondral defect. This review is aimed at highlighting recent progress in utilizing bioengineering approaches to improve stem cell therapies for osteochondral diseases, which include microgel encapsulation, adhesive bioinks, and bioprinting to control the administration and distribution. We will also explore utilizing synthetic biology tools to control the differentiation fate and deliver therapeutic biomolecules to modulate the immune response. Finally, future directions and opportunities in the development of more potent and predictable stem cell therapies for osteochondral repair are discussed.

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Vγ9Vδ2 T cells as a promising innovative tool for immunotherapy of hematologic malignancies

Oncology Reviews ◽

10.4081/oncol.2010.211 ◽

2011 ◽

Vol 4 (4) ◽

pp. 211

Author(s):

Serena Meraviglia ◽

Carmela La Mendola ◽

Valentina Orlando ◽

Francesco Scarpa ◽

Giuseppe Cicero ◽

...

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Ex Vivo ◽

Γδ T Cells ◽

Hematologic Malignancies ◽

Cytolytic Activity ◽

Cell Therapies ◽

Stressed Cells

The potent anti-tumor activities of γδ T cells, their ability to produce pro-inflammatory cytokines, and their strong cytolytic activity have prompted the development of protocols in which γδ agonists or ex vivo-expanded γδ cells are administered to tumor patients. γδ T cells can be selectively activated by either synthetic phosphoantigens or by drugs that enhance their accumulation into stressed cells as aminobisphosphonates, thus offering new avenues for the development of γδ T cell-based immunotherapies. The recent development of small drugs selectively activating Vγ9Vδ2 T lymphocytes, which upregulate the endogenous phosphoantigens, has enabled the investigators to design the experimental approaches of cancer immunotherapies; several ongoing phase I and II clinical trials are focused on the role of the direct bioactivity of drugs and of adoptive cell therapies involving phosphoantigen- or aminobisphosphonate-activated Vγ9Vδ2 T lymphocytes in humans. In this review, we focus on the recent advances in the activation/expansion of γδ T cells in vitro and in vivo that may represent a promising target for the design of novel and highly innovative immunotherapy in patients with hematologic malignancies.<br />

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Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

Journal of Experimental Medicine ◽

10.1084/jem.20160576 ◽

2016 ◽

Vol 213 (11) ◽

pp. 2413-2435 ◽

Cited By ~ 53

Author(s):

Yi Wang ◽

Cindy S. Ma ◽

Yun Ling ◽

Aziz Bousfiha ◽

Yildiz Camcioglu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Ex Vivo ◽

Cell Phenotype ◽

Loss Of Function ◽

Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

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Abstract 1767: Feasibility of In Vivo Cardiac Stem Cell Tracking, by SPECT and PET Imaging, Using the Sodium-iodide Symporter as Reporter Gene

Circulation ◽

10.1161/circ.116.suppl_16.ii_374 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

John Terrovitis ◽

Keng Fai Kwok ◽

Riikka Läutamaki ◽

James M Engles ◽

Andreas S Barth ◽

...

Keyword(s):

Stem Cell ◽

Reporter Gene ◽

Cell Tracking ◽

Ex Vivo ◽

Small Animal ◽

Cell Labeling ◽

Sodium Iodide Symporter ◽

Cell Injection

Background. Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo . Aim. To develop a reporter gene that permits in vivo stem cell labeling. We examined the sodium-iodide symporter (NIS), a protein that is not expressed in the heart, but promotes cellular uptake of 99m Tc or 124 I, thus permitting cell tracking by SPECT or PET imaging, respectively. Methods. The human NIS gene ( h NIS) was expressed in rat cardiac derived stem cells (rCDCs) using lentivirus driven by the CAG or CMV promoter. NIS function in transduced cells was confirmed by in vitro 99m Tc uptake. Eleven rats were injected with 1 or 2 million rCDCs intramyocardially immediately after LAD ligation; 6 with CMV-NIS and 5 with CAG-NIS cells. Dual isotope SPECT imaging was performed on a small animal SPECT/CT system, using 99m Tc for cell detection and 201 Tl for myocardial delineation, 24 hrs after cell injection. PET was performed on a small animal PET scanner using 124 I for cell tracking and 13 NH 3 for myocardial delineation, 48hrs after cell injection. Contrast Ratio (CR) was defined as [(signal in the cells)-(signal in blood pool)]/signal in blood pool. High resolution ex vivo SPECT scans of explanted hearts (n=3) were obtained to confirm that in vivo signal was derived from the cell injection site. The presence of h NIS mRNA was confirmed in injected hearts after animal sacrifice (n=2), by real-time RT-PCR. Results. NIS expression in rCDCs did not affect cell viability/proliferation (p=0.718, ctr vs NIS). In vitro 99m Tc uptake was 6.0±0.9% vs 0.07±0.05, without and with perchlorate (specific NIS blocker), respectively. NIS-transduced rCDCs were easily visualized as spots of 99m Tc or 124 I uptake within a perfusion deficit in the SPECT and PET images. CR was considerably higher when cells were transduced by the CMV-NIS virus in comparison to the CAG-NIS virus (70±40% vs 28±29%, p=0.085). Ex vivo small animal SPECT imaging confirmed that in vivo 99m Tc signals were localized to the injection sites. PCR confirmed the presence of h NIS mRNA in injected hearts. Conclusion. NIS expression allows non invasive in vivo stem cell tracking in the myocardium, using both SPECT and PET. This reporter gene has great potential for translation in future clinical applications.

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Dose-Independent Therapeutic Benefit of Bone Marrow Stem Cell Transplantation after MI in Mice

Biomedicines ◽

10.3390/biomedicines8060157 ◽

2020 ◽

Vol 8 (6) ◽

pp. 157

Author(s):

Nicole Zarniko ◽

Anna Skorska ◽

Gustav Steinhoff ◽

Robert David ◽

Ralf Gaebel

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Ex Vivo ◽

Therapeutic Benefit ◽

Dependent Manner ◽

Mesenchymal Progenitors

Several cell populations derived from bone marrow (BM) have been shown to possess cardiac regenerative potential. Among these are freshly isolated CD133+ hematopoietic as well as culture-expanded mesenchymal stem cells. Alternatively, by purifying CD271+ cells from BM, mesenchymal progenitors can be enriched without an ex vivo cultivation. With regard to the limited available number of freshly isolated BM-derived stem cells, the effect of the dosage on the therapeutic efficiency is of particular interest. Therefore, in the present pre-clinical study, we investigated human BM-derived CD133+ and CD271+ stem cells for their cardiac regenerative potential three weeks post-myocardial infarction (MI) in a dose-dependent manner. The improvement of the hemodynamic function as well as cardiac remodeling showed no therapeutic difference after the transplantation of both 100,000 and 500,000 stem cells. Therefore, beneficial stem cell transplantation post-MI is widely independent of the cell dose and detrimental stem cell amplification in vitro can likely be avoided.

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