Source Of Pluripotent Stem Cells Research Articles

Articular Cartilage Injury; Current Status and Future Direction.

Today, treatments of cartilage and osteochondral lesions are routine clinical procedures. The avascular and hard-to-self-repair nature of cartilage tissue has posed a clinical challenge for the replacement and reconstruction of damaged cartilage. Treatment of large articular cartilage defects is technically difficult and complex, often accompanied by failure. Articular cartilage cannot repair itself after injury due to a lack of blood vessels, lymph, and nerves. Various treatments for cartilage regeneration have shown encouraging results, but unfortunately, none have been the perfect solution. New minimally invasive and effective techniques are being developed. The development of tissue engineering technology has created hope for articular cartilage reconstruction. This technology mainly supplies stem cells with various sources of pluripotent and mesenchymal stem cells. This article describes the treatments in detail, including types, grades of cartilage lesions, and immune mechanisms in cartilage injuries.

The Evolutionary Route of in vitro Human Spermatogenesis: What is the Next Destination?

Malfunction in spermatogenesis due to genetic diseases, trauma, congenital disorders or gonadotoxic treatments results in infertility in approximately 7% of males. The behavior of spermatogonial stem cells (SSCs) within three-dimensional, multifactorial, and dynamic microenvironment implicates a niche that serves as a repository for fertility, since can serve as a source of mature and functional male germ cells. Current protocols enable reprogramming of mature somatic cells into induced pluripotent stem cells (iPSCs) and their limited differentiation to SSCs within the range of 0-5%. However, the resulting human iPSC-derived haploid spermatogenic germ cell yield in terms of number and functionality is currently insufficient for transfer to infertility clinic as a therapeutic tool. In this article, we reviewed the evolution of experimental culture platforms and introduced a novel iPSCs-based approach for in vitro spermatogenesis based on a niche perspective bearing cellular, chemical, and physical factors that provide the complex arrangement of testicular seminiferous tubules embedded within a vascularized stroma. We believe that bioengineered organoids supported by smart bio-printed tubules and microfluidic organ-on-a-chip systems offer efficient, precise, personalized platforms for autologous pluripotent stem cell sources to undergo the spermatogenetic cycle, presenting a promising tool for infertile male patients with complete testicular aplasia.

A Narrative Review on the Management of Severe COVID-19 Infection Using Stem Cell-based therapies with a Focus on the Registered Clinical Trials

The emergence of SARS-CoV-2 has led to a concerning global pandemic. The severity of COVID-19 symptoms may be enhanced due to underlying medical conditions. Several studies demonstrated severe COVID-19 infection can lead to innate and adaptive immune dysregulation, cytokine storms as well and the formation of fibromyxoid exudate in the respiratory alveolar, ultimately resulting in pulmonary fibrosis and ARDS as the leading cause of mortality and morbidity. Currently, there is a widespread global endeavor in finding efficient drugs or vaccines to manage COVID-19. Although some FDA-approved treatments have been introduced for COVID-19, alternative therapies might decrease the mortality rates. Various sources of pluripotent and mesenchymal stem cells as cell-based therapies have been applied on moderate to severe COVID-19 patients with acute respiratory distress syndrome, leading to positive results. Cell-based therapies by modulating the cytokine cascades and cellular apoptosis can probably inhibit tissue remodeling and subsequent end-organ damage. The present review aims to discuss the advantages of stem cell-based therapies in the treatment of COVID-19 patients and the possible challenges associated with their application.

Integration-free induced pluripotent stem cells from three endangered Southeast Asian non-human primate species

Advanced molecular and cellular technologies provide promising tools for wildlife and biodiversity conservation. Induced pluripotent stem cell (iPSC) technology offers an easily accessible and infinite source of pluripotent stem cells, and have been derived from many threatened wildlife species. This paper describes the first successful integration-free reprogramming of adult somatic cells to iPSCs, and their differentiation, from three endangered Southeast Asian primates: the Celebes Crested Macaque (Macaca nigra), the Lar Gibbon (Hylobates lar), and the Siamang (Symphalangus syndactylus). iPSCs were also generated from the Proboscis Monkey (Nasalis larvatus). Differences in mechanisms could elicit new discoveries regarding primate evolution and development. iPSCs from endangered species provides a safety net in conservation efforts and allows for sustainable sampling for research and conservation, all while providing a platform for the development of further in vitro models of disease.

Bone marrow aspirate concentrate – A novel approach to alter the course of temporomandibular joint osteoarthritis (a clinical study)

IntroductionTMJ OA is characterized by severe osteocartilaginous degradation of the joint structure resulting in severe deterioration of both joint function as well as joint structure. bone marrow aspirate concentrate (BMAC) gained wide acceptance as an auspicious addition for regenerative medicine as it is confirmed to be a rich source of pluripotent mesenchymal stem cells and growth factors that produce promising relief of clinical symptoms with significant repair of the joint structure. Thus, the study aims at assessing the efficacy of bone marrow aspirate concentrate (BMAC) as a treatment modality for TMJ osteoarthritis and compare its efficacy with that of hyaluronic acid (HA). Methods24 patients were included in the present study and divided into 12 patients in each group. Joint arthrocentesis was performed to all patients followed by intra-articular BMAC injection in Group I. While Group II received HA acid injection ResultsA trend towards long term joint repair at 12 and 18 months follow up period was observed in the bone marrow aspirate concentrate (BMAC) group as a therapeutic modality for TMJ OA by providing necessary growth factors and anti-inflammatories that impedes the progression of the osteoarthritic degeneration. On the contrary to the viscosupplementary action of hyaluronic acid (HA) that showed relapse of patients conditions. ConclusionBone marrow aspirate concentrate (BMAC) is able to reverse the degenerative effects of TMJ OA however,further studies are mandatory with larger population and longer follow-up time.

Transformation of Pluripotency States during Morphogenesis of Mouse and Human Epiblast

The pluripotent status of a cell in vivo is spatio-temporally regulated within embryogenesis and is determined by the processes of self-renewal, endless proliferation and differentiation into all cell types of the body. Previously, the pluripotency was characterized using teratocarcinoma cells. Then this term was applied to the embryonic cells of the preimplantation mouse embryo. Preimplantationally formed mouse and human pluripotent stem cells (PSCs) appear to exist until gastrulation. One of the main events in the early mammalian development is the differentiation of the inner cell mass of the blastocyst (ICM) into a hypoblast and an epiblast, which develops into the embryo itself. Continuous and dynamic transformation of pluripotency states in development coincides with the morphogenetic processes, which are involved in the formation and maturation of the epiblast. Thus, blastocyst ICM cells differ in epigenetic and transcription patterns from their daughter cells forming the peri/post-implantation epiblast. With the onset of gastrulation movements, the maturation of epiblast cells ends with their differentiation into cells of three germ layers. This review considers the historical aspects of the study of cell pluripotency, various sources of PSCs, mechanisms and signaling pathways that support self-renewal and pluripotency in PSC cultures. In addition, we summarize and conceptualize data on morphogenetic processes that are involved in the formation of naive ICM cells in vivo and the subsequent maturation of mouse and human epiblast cells associated with the transformation of their pluripotency states.

Transcriptomic analysis of feeder-free culture system for maintaining naïve-state pluripotency in human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (PSCs) have the capacity of self-renewal and multilineage differentiation in vitro. Conventional hPSCs, which are in a primed state, can produce various types of differentiated cells. However, the variability in their degree of pluripotency and differentiation propensities, which is influenced by the inductive methods and culture conditions, limit their availability. Therefore, PSCs in a naïve state are a promising source of PSCs. We recently developed a culture system for naïve hPSCs using an inhibitor of the NOTCH signaling pathway and a histone H3 methyltransferase disruptor. This culture system requires feeder cells for stably maintaining the naïve hPSCs. We aimed to develop a culture system for hPSCs that could maintain pluripotency under feeder-free conditions. We used two inhibitors to develop an alternative feeder-free culture system to obtain naïve hPSCs. The naïve cells underwent stable cellular proliferation and were positive for naïve stem cell markers; in addition, they could differentiate into the three germ layers. These feeder-free dome-shaped induced pluripotent stem cells (FFDS-iPSCs) have characteristics similar to that of naïve-like PSCs. The naive hPSCs under feeder-free conditions could ensure supply of cells for various applications in regenerative medicine and disease modeling.

Clinical evaluation of periosteal pedicle flap in the treatment of gingival recessions for esthetic root coverage: A randomized controlled clinical trial.

Root coverage using subepithelial connective tissue graft (SCTG) is gold standard with few drawbacks such as requirement of second surgical site, reduced donor tissue availability, and increased surgical complications and pain. Periosteal pedicle graft (PPG) due to its rich source of pluripotent stem cells and nonrequirement of second surgical site could be a successful alternative for invasive SCTG. Therefore, current research aims to compare the amount of root coverage obtained with PPG to SCTG. Fifty-two single gingival recessions were included in the sample, with 26 patients allocated to the SCTG (control) and PPG (test) groups at random. At baseline, 3, and 6 months after surgery, clinical measurements of probing depth, clinical attachment level (CAL), recession depth (RD), recession width (RW), and width of keratinized tissue were taken. Both procedures, i.e., SCTG and PPG, resulted in root coverage to a variable extent and a significant decrease in RD (1.69, 1.38 mm in SCTG and PPG groups, respectively) and RW, CAL gain is found in both the groups without any intergroup statistical variance. Complete root coverage (CRC) was obtained in 14 out of 26, i.e., 53.8% defects in both SCTG and PPG groups. A better comfort level is found in the group treated with PPG. Gingival recessions can be successfully treated with PPG, which has predictability comparable to SCTG and does not require a second surgical site.

Subretinal Transplant of Human Amniotic Membrane in Advanced Age-Related Macular Degeneration.

Macular neovascularization (MNV) and geographic atrophy can complicate age-related macular degeneration (AMD) and lead to severe visual acuity reduction. Despite the medical treatments available, with a defect in the retinal pigmented epithelium (RPE) there is no possibility of restoring acceptable visual acuity. We evaluated postoperative outcomes in patients affected by advanced AMD who underwent subretinal implant of the human amniotic membrane (hAM) as a source of pluripotent stem cells. This retrospective, consecutive, non-randomized interventional study included 23 eyes of 21 patients affected by AMD complicated by MNV, and five eyes of five patients affected by geographic atrophy. All eyes underwent a pars plana vitrectomy, neovascular membrane removal for the MNV group, a subretinal implant of hAM, and gas tamponade, and were followed for 12 months. The primary study outcome was visual acuity improvement. Secondary outcomes were postoperative complications, OCT-angiography parameters correlated with best-corrected visual acuity (BCVA) and MNV recurrence. The mean preoperative BCVA was 1.9 logMAR, and the mean final BCVA value was 1.2 logMAR. In the MNV group, the mean BCVA improved from 1.84 logMAR to 1.26 logMAR, and from 1.84 logMAR to 1.32 logMAR in the geographic atrophy group. No MNV recurrence was evident in 12 months of follow-up. An OCT-angiography scan was used to evaluate the retinal vascularization in the treated eye, which showed a high correlation between BCVA and deep vascular density. This study demonstrates the hAM potential and safety in promoting a partial restoration of retinal function together with an increase in visual acuity.

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

Abstract 13139: Single Gene Cardiopoiesis Induced in Human Pluripotent Source

Introduction: The process of engaging stem cells towards a cardiopoietic (CP) state was established using a protein-based cocktail consisting of multiple growth factors. Although this regenerative enhancement protocol has been successfully tested in advanced clinical trials, it remains resource-intensive. Recent efforts have focused on streamlining cardiopoiesis through a protein-independent alternative that leverages a transfection-based mRNA approach. Microencapsulated modified messenger RNA (M3RNA) was utilized to deliver brachyury (T), an early mesodermal transcription factor to provoke early lineage specification in pluripotent cells. Hypothesis: Transfection of a human pluripotent stem cell source with T induces early expression of CP transcription factors to accelerate cardiac differentiation. Methods: Human pluripotent stem cells were transfected with T and maintained for 14 days. Expression of the cardiopoietic markers MEF2C, GATA4, and Nkx2.5 was analyzed using RT-qPCR, in tandem with MYH6 at days 10 and 20. Differentiation proficiency was independently corroborated by assessing Mef2c and Nkx2.5 expression by IF. Results: Following transfection with T, Nkx2.5, MEF2C and GATA4 were significantly induced at 72h (60-, 20- and 40-fold, respectively, p<0.01 for all 3 markers vs baseline), reflecting timing of differentiation initiation. At 5 days post differentiation, expression of the cardiac-specific Nkx2.5 increased by 5 fold vs control and the cell cycle regulator CHIR (p<0.01), suggesting successful persistent induction of cardiopoeisis. At 10 and 20 days of differentiation, RNA expression of MYH6 was 10-15 times induced in human pluripotent cells transduced with T (p < 0.0001, and p < 0.001 vs control, respectively), compared to 2 times (p<0.05, p<0.01 vs control, respectively) in CHIR treated cohorts. Accordingly, at 42 days post-induction of differentiation, a greater number of cells expressed MLC2V and cTnT (p<1e-05, <1e-07, respectively vs control), driving a robust beating pattern. Conclusion: This study implements a single gene T M3RNA-based approach to readily induce cardiac differentiation from pluripotent sources, exceeding available commercial platforms.

Single-Cell Approaches to Deconvolute the Development of HSCs

Hematopoietic stem cells (HSCs) play a core role in blood development. The ability to efficiently produce HSCs from various pluripotent stem cell sources is the Holy Grail in the hematology field. However, in vitro or in vivo HSC production remains low, which may be attributable to the lack of understanding of hematopoiesis. Here, we review the recent progress in this area and introduce advanced technologies, such as single-cell RNA-seq, spatial transcriptomics, and molecular barcoding, which may help to acquire missing information about HSC generation. We finally discuss unresolved questions, the answers to which may be conducive to HSC production, providing a promising path toward HSC-based immunotherapies.

Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties.

Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications.

Preparation of Absorption-Resistant Hard Tissue Using Dental Pulp-Derived Cells and Honeycomb Tricalcium Phosphate.

In recent years, there has been increasing interest in the treatment of bone defects using undifferentiated mesenchymal stem cells (MSCs) in vivo. Recently, dental pulp has been proposed as a promising source of pluripotent mesenchymal stem cells (MSCs), which can be used in various clinical applications. Dentin is the hard tissue that makes up teeth, and has the same composition and strength as bone. However, unlike bone, dentin is usually not remodeled under physiological conditions. Here, we generated odontoblast-like cells from mouse dental pulp stem cells and combined them with honeycomb tricalcium phosphate (TCP) with a 300 μm hole to create bone-like tissue under the skin of mice. The bone-like hard tissue produced in this study was different from bone tissue, i.e., was not resorbed by osteoclasts and was less easily absorbed than the bone tissue. It has been suggested that hard tissue-forming cells induced from dental pulp do not have the ability to induce osteoclast differentiation. Therefore, the newly created bone-like hard tissue has high potential for absorption-resistant hard tissue repair and regeneration procedures.

Sequential regulation of hemogenic fate and hematopoietic stem and progenitor cell formation from arterial endothelium by Ezh1/2.

SummaryAcross species, hematopoietic stem and progenitor cells (HSPCs) arise during embryogenesis from a specialized arterial population, termed hemogenic endothelium. Here, we describe a mechanistic role for the epigenetic regulator, Enhancer of zeste homolog-1 (Ezh1), in vertebrate HSPC production via regulation of hemogenic commitment. Loss of ezh1 in zebrafish embryos favored acquisition of hemogenic (gata2b) and HSPC (runx1) fate at the expense of the arterial program (ephrinb2a, dll4). In contrast, ezh1 overexpression blocked hematopoietic progression via maintenance of arterial gene expression. The related Polycomb group subunit, Ezh2, functioned in a non-redundant, sequential manner, whereby inhibition had no impact on arterial identity, but was capable of blocking ezh1-knockdown-associated HSPC expansion. Single-cell RNA sequencing across ezh1 genotypes revealed a dropout of ezh1+/− cells among arterial endothelium associated with positive regulation of gene transcription. Exploitation of Ezh1/2 modulation has potential functional relevance for improving in vitro HSPC differentiation from induced pluripotent stem cell sources.

Human Amniotic Epithelial Cells as a Tool to Investigate the Effects of Cyanidin 3-O-Glucoside on Cell Differentiation.

Cyanidin, a kind of anthocyanin, has been reported to have chemotherapeutic activities in humans. Human amniotic epithelial cells (hAECs) are considered a potential source of pluripotent stem cells. hAECs have been used as a novel tool in regenerative cellular therapy and cell differentiation studies. In this study, to explore the effects of cyanidin-3-O-glucoside (Cy3G) on hAECs and their mechanisms, we investigated the transcriptomic changes in the Cy3G-treated cells using microarray analysis. Among the differentially expressed genes (Fold change > 1.1; p-value < 0.05), 109 genes were upregulated and 232 were downregulated. Ratios of upregulated and downregulated genes were 0.22% and 0.47% of the total expressed genes, respectively. Next, we explored the enriched gene ontology, i.e., the biological process, molecular function, and cellular component of the 37 upregulated (>1.3-fold change) and 124 downregulated (<1.3-fold change) genes. Significantly enriched biological processes by the upregulated genes included “response to muscle activity,” and the genes involved in this gene ontology (GO) were Metrnl and SRD5A1, which function in the adipocyte. On the other hand, the cell cycle biological process was significantly enriched by the downregulated genes, including some from the SMC gene family. An adipogenesis-associated gene DDX6 was also included in the cell cycle biological process. Thus, our findings suggest the prospects of Cy3G in modulating adipocyte differentiation in hAECs.

Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells: Current Controversies.

Brain microvascular endothelial cells (BMECs) possess unique properties that are crucial for many functions of the blood-brain-barrier (BBB) including maintenance of brain homeostasis and regulation of interactions between the brain and immune system. The generation of a pure population of putative brain microvascular endothelial cells from human pluripotent stem cell sources (iBMECs) has been described to meet the need for reliable and reproducible brain endothelial cells in vitro. Human pluripotent stem cells (hPSCs), embryonic or induced, can be differentiated into large quantities of specialized cells in order to study development and model disease. These hPSC-derived iBMECs display endothelial-like properties, such as tube formation and low-density lipoprotein uptake, high transendothelial electrical resistance (TEER), and barrier-like efflux transporter activities. Over time, the de novo generation of an organotypic endothelial cell from hPSCs has aroused controversies. This perspective article highlights the developments made in the field of hPSC derived brain endothelial cells as well as where experimental data are lacking, and what concerns have emerged since their initial description.

Update on the role of emerging stem cell technology in head and neck medicine.

Head and neck surgery is a broad discipline that involves the management of complex conditions such as burns, skin cancer, head and neck cancer, congenital abnormalities, and facial rejuvenation. For patients with cancer, surgery, radiotherapy, and chemotherapy are often the main modes of treatment. Many patients require follow-up reconstructive surgery, and the use of stem cells offers novel treatments that could aid recovery. Laryngeal, tracheal, and neuronal tissues are frequently damaged by surgery in the head and neck and these tissues have little intrinsic regenerative ability. Pluripotent embryonic stem cells retain the ability to differentiate into a wide variety of cells meaning that large tissue defects can be reduced by stimulating new cell growth. Research has demonstrated potential benefits of using stem cells in facial rejuvenation procedures and the management of burns sequelae. The advancements made in the use of adult progenitor stem cells as a possible source for pluripotent stem cells (induced pluripotent stem cells) mean that ethical considerations around the use of embryological tissue can be minimized, allowing for more research to take place. Currently, the evidence base for the use of stem cells in head and neck surgery is limited, but it has now been proven that stem cells can act as a source for lost or damaged tissue in the head and neck. With continuous advancements being made in the fields of tissue engineering, it is likely that stem cells will play a major role in head and neck surgery in the future.

Steps toward the generation of ovarian somatic cells from pluripotent stem cells

The generation of functional gametes, both eggs and sperm, from murine pluripotent stem cell (PSC) sources, has set the stage for the eventual use of this emerging technology in other species. With the field enthusiastically embracing this eventuality, in particular for animal conservation efforts, there are a number of key factors to consider regarding the applicability of these methods across species, particularly with regard to the generation of eggs. To date, published studies point to the need for fetal somatic tissue and primitive granulosa cells to serve as a niche for the growth and maturation of oocytes generated from PSCs. In practice, the need for such tissue represents a major limitation when attempting to apply this to species in which access to fetal ovaries is limited or unethical. To circumvent this, we and others have derived methods to generate ovarian granulosa cells from PSCs, albeit with low yield. Herein we present an update on the status of generating early stage granulosa cells from PSCs, and provide evidence for improvements based on a stepwise, 2-dimensional protocol for the directed differentiation of human PSCs.

Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived-From the Same Induced Pluripotent Stem Cell Source.

In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.