Feeder Layer Research Articles

Cumulus cells secrete micro-vesicles carrying miRNAs: their role in the acquisition of mouse and human oocyte's developmental competence

Background and Aim: During folliculogenesis oocytes acquire competence also thanks to a bidirectional communication with companion cumulus cells (CCs). The nature of the CC-determinants contributing to this remains unclear. We recently showed that when mouse CCs-free antral oocytes are cultured to MII with a feeder layer (FL) of CCs isolated from developmentally-competent surrounded-nucleolus (FL-SN-CCs) or -incompetent not-surrounded-nucleolus (FL-NSN-CCs) oocytes, the former acquire developmental competence to blastocyst, whereas the latter arrest.

Different transcriptional profiles of human embryonic stem cells grown in a feeder-free culture system and on human foreskin fibroblast feeder layers.

Feeder cells provide an optimal microenvironment for the propagation of human embryonic stem cells (hESCs) by supplying currently known or unknown factors. However, the hESCs grown on feeder cells are not suitable for the purpose of clinical application because of the risk of contamination. In recent years, the feeder-free culture method has been developed to eliminate contamination, but some studies show that hESCs exhibit poor growth patterns in a feeder-free culture system. Regarding this phenomenon, we speculate that some genes related to hESC propagation were differently expressed in hESCs grown on feeder cells. To test this hypothesis, 3 hESC lines (NF4, NF5 and P096) were efficiently expanded in a feeder-free culture system or on human foreskin fibroblast (HFF) cells. The different gene expression patterns of hESCs in these 2 conditions were analyzed through microarrays. The results revealed that the hESCs cultured in both conditions maintained the expression of stemness markers and the ability to spontaneously differentiate into the 3 germ layers. The analysis of gene expression profiles revealed that 23 lncRNA and 15 genes were significantly differentially expressed in these two culture conditions. Furthermore, GO analyses showed that these genes were involved in such biological processes as growth factor stimuli, cell growth, and stem cell maintenance. To summarize, our study demonstrated that the hESCs grown on the HFF showed different gene expression patterns compared to those grown in a feeder-free culture system, suggesting that these differently expressed lncRNAs and genes played important roles in maintaining hESC propagation.

Bio-engineering of 3-D cell sheets for diabetic rats: Interaction between mesenchymal stem cells and beta cells in functional islet regeneration system

Type 1 diabetes is an autoimmune disease that emerges with the destruction of beta cells of pancreatic Langerhans islets. Three different therapeutical approaches have been developed so far; pancreas transplantation, islet transplantation, and cell-based therapies. Bioengineering cell sheets for tissue generating is one of the latest approaches that have been used to construct cell-sheets instead of single cells so that it mimics the in vivo environments more. In this study, extra-hepatic functional islet tissue was constructed by transferring the 3-D beta cells and GFP labelled MSCs MSC sheets to the subcutaneous site of rats with STZ-induced diabetes. rBM-MSCs and beta cells were cultured on the 6-well PIPAAm culture dishes. Obtained rBM-MSCs as two-cell sheets and beta cells cultured in droplets with matrigel has transplanted into the dorsal subcutaneous area of diabetic rats. Fasting blood glucose levels and body weights were evaluated for 30 days after transplantation. Immunocytochemistry analysis for the anti-apoptotic, anti-inflammatory, and angiogenetic effects of MSCs on the 30th day of subcutaneous cell transplantation. All recipient rats transplanted with beta-cells with MSCs returned toward normoglycemia by day 5 and remained at this level for 30 days. Immunocytochemical analyses supported that the MSCs and beta cells preserved their viability and function. MSCs secrete cytokines and growth factors TGF-β and IL-6; MSCs of the important features of the anti-apoptotic and anti-inflammatory properties, thanks to apoptosis of beta cells preserve graft explained by inhibition. In transplantation of MSCs induced angiogenesis and neovascularization, PECAM-1 and GFP positive simultaneously determining endothelial cells was observed indicating. Subcutaneous 3D beta-cell transplantation would be possible with the MSC-sheets as a feeder layer of beta cells. The beta-cell line is glucose-sensitive and has a high insulin release potential, and can be used as an alternative to islets in in vivo transplant studies.

Melt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork.

The permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure–function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125–500 μm and fiber diameters of 10–12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6–360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8–14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.

Endothelial-Smooth Muscle Cell Interactions in a Shear-Exposed Intimal Hyperplasia on-a-Dish Model to Evaluate Therapeutic Strategies.

Intimal hyperplasia (IH) represents a major challenge following cardiovascular interventions. While mechanisms are poorly understood, the inefficient preventive methods incentivize the search for novel therapies. A vessel‐on‐a‐dish platform is presented, consisting of direct‐contact cocultures with human primary endothelial cells (ECs) and smooth muscle cells (SMCs) exposed to both laminar pulsatile and disturbed flow on an orbital shaker. With contractile SMCs sitting below a confluent EC layer, a model that successfully replicates the architecture of a quiescent vessel wall is created. In the novel IH model, ECs are seeded on synthetic SMCs at low density, mimicking reendothelization after vascular injury. Over 3 days of coculture, ECs transition from a network conformation to confluent 2D islands, as promoted by pulsatile flow, resulting in a “defected” EC monolayer. In defected regions, SMCs incorporated plasma fibronectin into fibers, increased proliferation, and formed multilayers, similarly to IH in vivo. These phenomena are inhibited under confluent EC layers, supporting therapeutic approaches that focus on endothelial regeneration rather than inhibiting proliferation, as illustrated in a proof‐of‐concept experiment with Paclitaxel. Thus, this in vitro system offers a new tool to study EC‐SMC communication in IH pathophysiology, while providing an easy‐to‐use translational disease model platform for low‐cost and high‐content therapeutic development.

Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C.

Feeder cells play important roles in In-vitro culture of stem cells. However, the preparation protocol of feeder cells produced by bovine embryonic fibroblast cells (bEFs) is still lack. In this study, the preparation of bEF-feeder by Mitomycin C was optimized with different concentrations and treatment time. The cell viability of bEFs was detected by CCK8 and 5-Ethynyl-2'-deoxyuridine. The growth of bESCs in each bEFs-feeder group was assessed by alkaline phosphatase staining and CCK8. Quantitative real time PCR was used to detect the mRNA expression of pluripotency-related genes of bESCs. Results showed that the proliferation of bEFs was significantly repressed while bEFs were treated with 14 ug/mL or 16 ug/mL Mitomycin C for 3h, and the cell viability within 2-4days after treatment was consistent with the 1st day. The numbers of bESCs clones in bEF-feeder treated with 14μg/mL Mitomycin C for 3h or 16μg/mL Mitomycin C for 3h were significantly higher than that in bEF-feeder treated with 8μg/mL Mitomycin C for 8h or bEFs treated with 6μg/mL Mitomycin C for 9h. The mRNA expression of pluripotency-related genes in bESCs cultured by bEF-feeder were higher than the MEF-feeder, the clone morphology of bESCs cultured in bEF-feeder was rounder and sharper than the MEF-feeder. In conclusion, the bEF-feeder prepared with 14μg/mL Mitomycin C for 3h or 16μg/mL Mitomycin C for 3h could effectively maintains the growth of bESCs, and bEF-feeder is more suitable for bESCs culture than the MEF-feeder.

The human amniotic epithelium confers a bias to differentiate toward the neuroectoderm lineage in human embryonic stem cells.

Human embryonic stem cells (hESCs) derive from the epiblast and have pluripotent potential. To maintain the conventional conditions of the pluripotent potential in an undifferentiated state, inactivated mouse embryonic fibroblast (iMEF) is used as a feeder layer. However, it has been suggested that hESC under this conventional condition (hESC-iMEF) is an artifact that does not correspond to the in vitro counterpart of the human epiblast. Our previous studies demonstrated the use of an alternative feeder layer of human amniotic epithelial cells (hAECs) to derive and maintain hESC. We wondered if the hESC-hAEC culture could represent a different pluripotent stage than that of naïve or primed conventional conditions, simulating the stage in which the amniotic epithelium derives from the epiblast during peri-implantation. Like the conventional primed hESC-iMEF, hESC-hAEC has the same levels of expression as the 'pluripotency core' and does not express markers of naïve pluripotency. However, it presents a downregulation of HOX genes and genes associated with the endoderm and mesoderm, and it exhibits an increase in the expression of ectoderm lineage genes, specifically in the anterior neuroectoderm. Transcriptome analysis showed in hESC-hAEC an upregulated signature of genes coding for transcription factors involved in neural induction and forebrain development, and the ability to differentiate into a neural lineage was superior in comparison with conventional hESC-iMEF. We propose that the interaction of hESC with hAEC confers hESC a biased potential that resembles the anteriorized epiblast, which is predisposed to form the neural ectoderm.

Electroporation Microchip With Integrated Conducting Polymer Electrode Array for Highly Sensitive Impedance Measurement.

Monitoring of impedance changes during electroporation-based treatments can be used to study the biological response and provide feedback regarding treatment progression. However, seamless integration of the sensing electrodes with the setup can be challenging and high impedance sensing electrodes limit the recording sensitivity as well as the spatial resolution. Here, we present an all-in-one microchip containing stimulation electrodes, as well as an array of low impedance, micro-scale sensing electrodes for highly sensitive impedance monitoring. An in vitro platform is fabricated with integrated stimulation and sensing electrodes. To reduce the impedance, the sensing electrodes are coated with the conducting polymer PEDOT:PSS. The performance is studied during the growth of a confluent cell layer and treatment with electrical pulses. Coated electrodes, compared to uncoated electrodes, show more pronounced impedance changes in a broader frequency range throughout the formation of a confluent cell layer and after electrical treatment. PEDOT:PSS coatings enhance the monitoring of impedance changes with micro-scale electrodes, enabling high spatial resolution and increased sensitivity. Such monitoring systems can be used to study electroporation dynamics and monitor treatment progression for better understanding of underlying mechanisms and improved outcomes.

Micro/nanoscale surface engineering to enhance hemocompatibility and reduce bacterial adhesion for cardiovascular implants

Precise engineering of surface properties is essential for improving bio/hemocompatibility of medical implants. In this study, potential of electrospraying (ES) as a coating method to control surface properties was evaluated in vitro. Poly (lactic acid) was coated on 316 L stainless steel plates by ES and spin-coating methods. Surface topography and morphology were studied using confocal and scanning electron microscope (SEM), respectively. Surface hydrophobicity and mechanical properties were investigated by contact angle and nanoindentation tests. Hemocompatibility of samples evaluated using protein adsorption, hemolysis, blood coagulation, complement system and leukocyte activation experiments. Platelet response to the coatings was examined using LDH assay, SEM and ELISA tests. Cell viability studies performed on human endothelial cells using MTT assay and SEM after 1, 4 and 7 days. SEM was used to consider formation of pseudomonas aeruginosa biofilms on the coatings. The results showed that ES increased hydrophobicity (122֯ vs 90֯) and surface roughness (2 μm vs 1.2 μm) but reduced surface stiffness (200 MPa vs 10800 MPa) in comparison to spin-coated samples. ES-coatings had microbead/nanofiber morphology while spin-coated samples had micro/nanoporous structure. Importantly, an almost confluent layer of endothelial cells was observed on ES-coated samples after 4 days in contrast to spin-coated ones. Various blood experiments revealed both coatings were hemocompatible and there was no significant difference among ES and spin-coated samples. Furthermore, ES-coated samples showed significantly lower propensity for biofilm formation. In conclusion, in vitro results revealed potential of ES to engineer different coating properties including hemo/biocompatibility and biofilm resistance for cardiovascular implants applications.

Electric Cell-Substrate Impedance Sensing (ECIS) as a Platform for Evaluating Barrier-Function Susceptibility and Damage from Pulmonary Atelectrauma.

Biophysical insults that either reduce barrier function (COVID-19, smoke inhalation, aspiration, and inflammation) or increase mechanical stress (surfactant dysfunction) make the lung more susceptible to atelectrauma. We investigate the susceptibility and time-dependent disruption of barrier function associated with pulmonary atelectrauma of epithelial cells that occurs in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). This in vitro study was performed using Electric Cell-substrate Impedance Sensing (ECIS) as a noninvasive evaluating technique for repetitive stress stimulus/response on monolayers of the human lung epithelial cell line NCI-H441. Atelectrauma was mimicked through recruitment/derecruitment (RD) of a semi-infinite air bubble to the fluid-occluded micro-channel. We show that a confluent monolayer with a high level of barrier function is nearly impervious to atelectrauma for hundreds of RD events. Nevertheless, barrier function is eventually diminished, and after a critical number of RD insults, the monolayer disintegrates exponentially. Confluent layers with lower initial barrier function are less resilient. These results indicate that the first line of defense from atelectrauma resides with intercellular binding. After disruption, the epithelial layer community protection is diminished and atelectrauma ensues. ECIS may provide a platform for identifying damaging stimuli, ventilation scenarios, or pharmaceuticals that can reduce susceptibility or enhance barrier-function recovery.

Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells

The pluripotency maintenance of pluripotent stem cells (PSCs) requires the suitable microenvironment, which commonly provided by feeder layers. However, the preparation of feeder layers is time consuming and labor exhaustive, and the feeder cells treated with mitomycin C or γ-ray irradiation bring heterologous contamination. In this study, mouse embryonic fibroblasts (MEFs) were treated by methanol to generate chemical fixed feeder cells, and bovine embryonic stem cells F7 (bESC-F7) cultured on this feeder layer. Then the pluripotency and metabolism of bESC-F7 cultured on methanol-fixed MEFs (MT-MEFs) named MT-F7 was compared with mitomycin C treated MEFs (MC-MEFs). The results showed that bESC-F7 formed alkaline phosphatase positive colonies on MT-MEFs, the relative expression of pluripotent markers of these cells was different from the bESCs cultured on the MC-MEFs (MC-F7). The long-term cultured MT-F7 formed embryoid bodies, showed the ability to differentiate into three germ layers similar to MC-F7. The analyses of RNA-seq data showed that MT-MEFs lead bESCs to novel steady expression patterns of genes regulating pluripotency and metabolism. Furthermore, the bovine expanded pluripotent stem cells (bEPSCs) cultured on MT-MEFs formed classical colonies, maintained pluripotency, and elevated metabolism. In conclusion, MT-MEFs were efficient feeder layer that maintain the distinctive pluripotency and metabolism of PSCs.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modulate Hematopoietic Stem and Progenitor Cell Viability and the Expression of Cell Cycle Regulators in an Age-dependent Manner

Aging of the hematopoietic system is characterized by an expansion of hematopoietic stem and progenitor cells (HSPCs) with reduced capacity for engraftment, self-renewal, and lymphoid differentiation, resulting in myeloid-biased hematopoiesis. This process is mediated by both HSPC intrinsic and extrinsic factors, e.g., the stromal environment. A relevant cellular component of the bone marrow (BM) microenvironment are mesenchymal stromal cells (MSCs) which regulate fate and differentiation of HSPCs. The bi-directional communication with HSPCs is mediated either by direct cell-cell contacts or by extracellular vesicles (EVs) which carry bioactive substances such as small RNA, DNA, lipids and proteins. So far, the impact of MSC-derived EVs on human hematopoietic aging is poorly investigated. BM MSCs were isolated from young (n = 3, median age: 22 years) and aged (n = 3, median age: 70 years) donors and the EVs were isolated after culturing the confluent cell layer in serum-free medium for 48 h. CD34+ HSPCs were purified from peripheral blood of healthy donors (n = 3, median age: 65 years) by magnetic sorting. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot detection of EV markers CD63, CD81 and Flotillin-1 revealed no significant differences between young and aged MSC-EVs. Interestingly, young MSCs secreted a significantly higher miRNA concentration than aged cells. However, the amount of distinct miRNAs such as miR-29a and miR-34a was significantly higher in aged MSC-EVs. HSPCs incubated with young EVs showed a significant increase in cell number and a higher viability. The expression of the tumor suppressors PTEN, a known target of mir-29a, and CDKN2A was increased in HSPCs incubated with young EVs. The clonogenic assay demonstrated a decreased colony number of CFU-GM after treatment with young EVs and an increased number of BFU-E/CFU-E after incubation with aged MSC-EVs. Xenogenic transplantation experiments showed no significant differences concerning the engraftment of lymphoid or myeloid cell compartments, but the overall human chimerism 8–16 weeks after transplantation was higher after EV treatment. In conclusion, our data suggest that HSPC characteristics such as cell cycle activity and clonogenicity can be modulated by MSC-derived EVs. Further studies have to elucidate the potential therapeutic relevance of our findings.

Human Oral Mucosal Fibroblasts from Limbal Stem Cell Deficient Patients as an Autologous Feeder Layer for Epithelial Cell Culture

Purpose To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use. Methods HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens–Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD. Results HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups. Conclusions HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

Expanding Homogeneous Culture of Human Primordial Germ Cell–Like Cells Maintaining Germline Features Without Serum or Feeder Layers

(Abstracted from Stem Cell Rep 2022;17(3):507–521) Primordial germ cells (PGCs) are embryonic precursors of gametes that are extensively studied in research settings. Human primordial PGC–like cells (hPGCLCs) are a surrogate model for embryonic PGCs that present difficulties in in vitro cell culture and expansion due to rapid loss of PGC-like identity and limited cell survival.

A Descriptive Study of the Physical Direct Interaction between Adipose Tissue-Mesenchymal Stem Cells and Colo 205 Cells: Impact on Cancer Cells Stemness, and Intracellular Reactive Oxygen Species Levels.

Background:Mesenchymal stem cells (MSCs) are widely used in clinical research to treat a wild spectrum of diseases due to their homing ability to damaged tissues, self-renewal capacity, and differentiation ability into various types of cells. In this research, we are describing the physical direct interaction between AT-MSCs and colon cancer cells, its impact on the stemness of colon cancer cells, along with the levels of intracellular Reactive Oxygen Species (ROS) levels in both types of cells. Methods:Adipose-tissue mesenchymal stem cells (AT-MSCs) were characterized by the means of MSCs classical markers expression using flow cytometry, and multilineage differentiation through osteogenic and adipogenic differentiation. MSCs and colo205 cells were cocultured in monolayer and 3D techniques in a ratio of 1:3 for 72 hours without media exchange and compared to monocultured cells. The physical direct interaction of cells in adhered culture and spheroids formation in ULA plates was observed using a light-inverted microscope. MSCs classical markers and cancer stem cells (CSCs) associated surface proteins were quantified in MSCs and colo205 cells. Intracellular ROS level was measured in both cell types. Surface protein and intracellular ROS quantification were carried out using flow cytometry. Results:CRC cells (colo205 cells) utilized MSCs as a feeder layer to grow and generate spheroids. The interaction increased the percentage of CSCs in colo205 population which was attributed to the increased expression of CD133, and reduced the levels of intracellular ROS in MSCs. Results indicated that MSCs support the growth, spheriod formation, and the stemness of colon cancer cells, while reducing the levels of intracellular ROS in MSCs.

The porcupine inhibitor WNT974 provokes ectodermal lineage differentiation of human embryonic stem cells.

Porcupine (Porcn) enzyme plays an essential role in Wnt signaling activation. Stearoyl-CoA desaturase-1 (SCD1) is required to provide Porcn substrates. The aim of this study was to determine the effect of a novel Porcn inhibitor on the fate of human embryonic stem cells (hESCs) and the reliance of Porcn on SCD1 activity. hESCs were cultured on a feeder layer or Matrigel-coated plates. Small molecules WNT974 (LGK-974) and CAY10566 were used to inhibit Porcn and SCD1 activity, respectively. We assessed the effect of Porcn inhibition on viability, expression of Wnt signaling targets, pluripotency markers, proliferation, differentiation, and protein fatty acylation. hESCs' conditioned medium (CM) containing secreted Wnt proteins were applied in rescue experiments. To examine the catalytic dependency of Porcn on SCD1, the results of combined inhibitor treatment were compared with the SCD1 inhibitor alone. LGK-974 at the selected concentrations showed mild effects on hESCs viability, but significantly reduced messenger RNA and protein expression of Wnt signaling targets (Axin-2 and c-Myc) and pluripotency markers (OCT-4 and SOX-2) (p < .05). Adding 1 μM of Porcn inhibitor reduced proliferation (p = .03) and enhanced differentiation capacity into ectodermal progenitors (p = .02), which were reverted by CM. Click chemistry reaction did not show significant alteration in protein fatty acylation upon LGK-974 treatment. Moreover, combined inhibitor treatment caused no further substantial reduction in Wnt signaling targets, pluripotency markers, and protein fatty acylation relative to CAY10566-treated cultures. The substrate availability for Porcn activity is regulated by SCD1 and targeting Porcn by LGK-974 prompts the transition of hESCs from self-renewal state to ectodermal lineage.

Long-term feeder-free culture of human pancreatic progenitors on fibronectin or matrix-free polymer potentiates β cell differentiation.

SummaryWith the aim of producing β cells for replacement therapies to treat diabetes, several protocols have been developed to differentiate human pluripotent stem cells to β cells via pancreatic progenitors. While in vivo pancreatic progenitors expand throughout development, the in vitro protocols have been designed to make these cells progress as fast as possible to β cells. Here, we report on a protocol enabling a long-term expansion of human pancreatic progenitors in a defined medium on fibronectin, in the absence of feeder layers. Moreover, through a screening of a polymer library we identify a polymer that can replace fibronectin. Our experiments, comparing expanded progenitors to directly differentiated progenitors, show that the expanded progenitors differentiate more efficiently into glucose-responsive β cells and produce fewer glucagon-expressing cells. The ability to expand progenitors under defined conditions and cryopreserve them will provide flexibility in research and therapeutic production.

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture.

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.

Directional capacity of human mesenchymal stem cells to support hematopoietic stem cell proliferation in vitro

Hematopoietic stem cells (HSCs) reside in a specialised microenvironment in the bone marrow, which is majorly composed of mesenchymal stem cells (MSCs) and its' derivatives. This study aimed to investigate the regulatory role of MSCs to decipher the cellular and humoral communications on HSCs' proliferation, self-renewal, and differentiation at the transcriptomic level. Microarray assay was employed to analyse the gene expression profile of HSCs that imparted by MSCs during co-culture. The proliferation of human umbilical cord blood-derived HSCs (hUC-HSCs) markedly propagated when MSCs were used as the feeder layer, without disturbing the undifferentiated state of HSCs, and reduced the cell death of HSCs. Upon co-culture with MSCs, the global microarray analysis of HSCs disclosed 712 differentially expressed genes (DEGs) (561 up-regulated and 151 down-regulated). The dysregulations of various transcripts were enriched for cellular functions such as cell cycle (including CCND1), apoptosis (including TNF), and genes related to signalling pathways governing self-renewal, as well as WNT5A from the Wnt signalling pathway, MAPK, Hedgehog, FGF2 from FGF, Jak-STAT, and PITX2 from the TGF-β signalling pathway. To concur this, real-time quantitative PCR (RT-qPCR) was utilised for corroborating the microarray results from five of the most dysregulated genes. This study elucidates the underlying mechanisms of the mitogenic influences of MSCs on the propagation of HSCs. The exploitation of such mechanisms provides a potential means for achieving larger quantities of HSCs in vitro, thus obviating the need for manipulating their differentiation potential for clinical application.

Cultivated Autologous Limbal Epithelial Cell Transplantation: New Frontier in the Treatment of Limbal Stem Cell Deficiency

Taking into consideration prior human experience with treating limbal stem cell deficiency (LSCD) with cultivated limbal epithelial cells (CLEC) from other countries, we have set a goal to optimize and standardize the techniques of CLEC preparation (called CALEC by our group) for the clinical trial in the United States. We performed an extensive literature review of all human trials, case series, and reports involving autologous cultivated limbal epithelial cell transplantation. Allogeneic cultivated limbal epithelial cell transplantations were reported only when combined with autologous studies. We also searched prior animal data aiding in detailing regulatory toxicology requirements. Between 1997 and 2020, the analysis of human trials revealed 21 studies on autologous grafts, and 13 studies analyzing both autologous grafts and allogeneic grafts. Of a total of 34 studies, 6 studies used good manufacturing process (GMP) facilities, and 11 studies had no animal-derived products or murine feeder layers, whereas only 1 study had both. Overall, the treatment with autologous CLEC grafts was 68.9% successful. In total there were 6 preclinical studies using rabbits, serving as surrogate studies to assess the safety and toxicity of cultivated limbal epithelial cells for human trials. Based on prior human experience, we further optimized the manufacturing conditions with GMP-grade and serum and animal-free reagents, and developed cell characterization assays for the CALEC product release. These data were used to develop a novel and consistent manufacturing process using only qualified and validated reagents for performing the first clinical trial on CALEC transplantation to treat LSCD in the United States.