JADICELL -Mesenchymal Stem Cells: A New Piece in the Puzzle of COVID-19 Treatment

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JADICELL - Burns Impair Blood-Brain Barrier and Mesenchymal Stem Cells Can Reverse the Process in Mice

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Burns Impair Blood-Brain Barrier and Mesenchymal Stem Cells Can Reverse the Process in Mice

https://www.frontiersin.org/articles/10 ... 78879/full

Neurological syndromes are observed in numerous patients who suffer burns, which add to the economic burden of societies and families. Recent studies have implied that blood–brain barrier (BBB) dysfunction is the key factor that induces these central nervous system (CNS) syndromes in peripheral traumatic disease, e.g., surgery and burns. However, the effect of burns on BBB and the underlying mechanism remains, largely, to be determined. The present study aimed to investigate the effect of burns on BBB and the potential of umbilical cord-derived mesenchymal stem cells (UC-MSCs), which have strong anti-inflammatory and repairing ability, to protect the integrity of BBB. BBB permeability was evaluated using dextran tracer (immunohistochemistry imaging and spectrophotometric quantification) and western blot, interleukin (IL)-6, and IL-1β levels in blood and brain were measured by enzyme-linked immunosorbent assay. Furthermore, transmission electron microscopy (TEM) was used to detect transcellular vesicular transport (transcytosis) in BBB. We found that burns increased mouse BBB permeability to both 10-kDa and 70-kDa dextran. IL-6 and IL-1β levels increased in peripheral blood and CNS after burns. In addition, burns decreased the level of tight junction proteins (TJs), including claudin-5, occludin, and ZO-1, which indicated increased BBB permeability due to paracellular pathway. Moreover, increased vesicular density after burns suggested increased transcytosis in brain microvascular endothelial cells. Finally, administering UC-MSCs at 1 h after burns effectively reversed these adverse effects and protected the integrity of BBB. These results suggest that burns increase BBB permeability through both paracellular pathway and transcytosis, the potential mechanism of which might be through increasing IL-6 and IL-1β levels and decreasing Mfsd2a level, and appropriate treatment with UC-MSCs can reverse these effects and protect the integrity of BBB after burns.

Introduction
Cognitive dysfunction caused by peripheral trauma has been frequently reported, especially postoperative cognitive dysfunction (1–3) and cognitive dysfunction after burns (4–12). These severe central nervous system (CNS) complications burden the patients’ families and the society. Increasing number of studies are focusing on CNS disorders caused by peripheral trauma. Our previous study in mice found that high concentrations of interleukin (IL)-6 in peripheral serum after abdominal surgery increased the permeability of blood–brain barrier (BBB) by disrupting tight junction proteins (TJs), and ultimately led to cognitive dysfunction (1). Danielson et al. found that in patients who underwent open heart surgery, peripheral systemic inflammation caused the dysfunction of the BBB (13). Above all, the pathological process of peripheral trauma that disrupts the integrity of BBB to affect CNS function has been widely accepted in medical field. Compared with the relatively simple incision of surgical trauma, burns cause more severe damage to the skin, elevating the levels of inflammatory factors and harmful substances in the serum. In case of patients with severe burns, serious CNS complications in addition to the damage done to their appearance, may strike the patients and even lead to the onset and progression of more serious psychological disorders. However, whether burns lead to BBB disruption and the underlying mechanisms involved remains to be determined.

BBB is the most important defensive barrier that protect the CNS from poisons and toxins (14–18). Most studies have suggested that its disruption caused by CNS diseases or injuries can exacerbate the development of diseases such as Alzheimer’s disease (19), multiple sclerosis (20), and traumatic brain injury (21). However, few studies have focused on BBB dysfunction caused by peripheral traumatic injury. The dysfunction of BBB leads to peripheral substances infiltrating into the CNS mainly via transcellular vesicular transport (transcytosis) and paracellular pathways (22). Our previous study suggested that the peripheral IL-6, a pro-inflammatory cytokine, decrease the level of TJs and thus induce the leakage of BBB to 10-kDa dextran, which may predicted the existence of paracellular pathway after abdominal surgery in mice and using anti-IL-6 neutralizing antibodies could effectively protect the integrity of BBB. However, burn trauma differs from common surgical trauma in two main aspects: First, burn trauma is considered more serious than peripheral surgical trauma, and the factors that induce BBB dysfunction after burns are still unknown. Therefore, use of anti-IL-6 antibodies is not possible. Second, burns belong to one of the most serious types of acute trauma whose occurrence is unpredictable. Therefore, the anti-inflammatory agents, which are limited in their application by the fact that they can be used only a few hours before traumatic injury, such as surgery, cannot be used for burns. Thus, new effective interventions are urgently required.

At present, mesenchymal stem cells (MSCs), which have powerful anti-inflammatory and wound repair effects, have a wide range of applications in medical field (23–26). In addition, some studies have found that MSCs can improve the progression of numerous neurological diseases, such as stroke (27–29), multiple sclerosis (30–32), and Alzheimer’s disease (33–35). Based on these reports, the potential role of MSCs in protecting the integrity of BBB after burn trauma needs to be further studied. MSCs have various strains derived from several tissues including bone marrow, adipose tissue, umbilical cord, placenta, and peripheral blood. Among these, MSCs derived from umbilical cord, also called umbilical cord MSCs (UC-MSCs), have stable biological characteristics, including anti-inflammatory effects and angiogenesis, and are relatively easy to obtain and abundantly available (36–39), making them a perfect choice for the present study. The present study aimed to assess the effect of burns on BBB permeability in animal models. We further aimed to investigate whether UC-MSCs could protect the integrity of BBB after burn trauma.

Materials and Methods
Umbilical Cord-Derived Mesenchymal Stem Cell Isolation and Culture
All animal experiments were conducted in accordance with the guidelines of the PLA General Hospital Standing Committee on the Use of Animals in Research and Teaching. One C57BL/6J pregnant mice (about 20 days), purchased from SPF Biotechnology Co., Ltd. (Beijing, China), was employed in this study and eight umbilical cords of which were used to isolate UC-MSCs. Briefly, umbilical cord tissues were harvested from these mice and dissected into small segments in which the veins and arteries were removed; 2 ml type I collagenase (Sigma Aldrich, St. Louis, MO, USA) was added to the tissues that were cultured in 60 mm plates and then were cultured at 37°C in 5% CO2 humidified atmosphere for 2 h. And then extracted the cell suspension in plates and placed it in centrifuge (1,000 r * 5 min). The suspension was abandoned and the cells was suspended with Dulbecco’s modified eagle medium (DMEM, Catalog: 2051855, Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) in 100 mm plates (Corning, NY, USA). The umbilical cord explants were cultured at 37°C in 5% CO2 humidified atmosphere, and the medium was changed every other day. UC-MSCs phenotype was assessed by flow cytometry based on the positivity for CD29, CD44, and Sca-1 and in the absence of CD31 and CD117 antigens (Cyagen, Santa Clara, CA, USA) (Figure S1 in Supplementary Material). Cells at passage 3 were used for treatment.

Discussion
Burns have been shown to induce cognitive impairment and abnormal behavior (4, 8–12). Given the fact that BBB dysfunction is the key factor in cognitive impairment (50–57) and that MSCs can effectively improve the integrity of BBB, we investigated whether burns could induce BBB dysfunction and neuroinflammation as well as evaluated its potential mechanism and the best time point for intervention to improve these effects after burn.

The sex of mice we have chosen for burn model was female. Although burn injury seem to be disproportionately an injury that occurs in males, female may be more vulnerable to the development of cognitive impairment after burn. Wasiak et al. found that in the 12 months post-injury, female patients showed overall poorer physical and mental health status, greater psychological distress, and greater difficulty with aspects of burn-specific health-related quality of life (HRQoL) (58). Other researches show that female after burn is more vulnerable to worse scar quality and mortality (59, 60). Therefore, we only used female mice in the current studies. Furthermore, because dextran-tracer is usually used to determine BBB integrity (61–68), we used it to estimate the effect of burn on BBB integrity in our research. We found that that burns increased the BBB permeability to 10-kDa as well as 70-kDa dextran, which is more difficult to infiltrate into CNS owing to its larger size. In addition, other studies have already demonstrated that the dosing of UC-MSCs (1 × 106) we have chosen is effective and safe. Pati et al. (69) have administered MSCs (1 × 106) for transplantation via tail vein at 2 and 24 h after traumatic brain injury in mice. Menge et al. (70) have administered MSCs (1 × 106) to transplant via tail vein at 2 and 24 h after traumatic brain injury in mice as well. Liu et al. (71) have administered MSCs (5 × 105) to transplant via tail vein at disease onset in EAE mice. Our current study has found that injecting UC-MSCs through tail veins of the mice at 0, 1, and 3 h after burn attenuated the burn-induced increase in BBB permeability. This suggested that UC-MSC treatment at 1 h after burn trauma could effectively improve the BBB permeability.

IL-6 and IL-1β are the main inflammatory cytokines that are released during inflammation, and their uncontrolled secretion can either induce or deteriorate the onset and progression of various diseases (1, 72–74). In addition, recent research found the overexpression of both cytokines was associated with the disruption of BBB. Uchida et al. (75) found that increasing IL-6 level in cerebrospinal fluid (CSF) of patients with neuromyelitis optica led to the disruption of BBB. Our previous research findings also confirmed that increasing IL-6 level in peripheral blood after abdominal surgery induced an increase in BBB permeability (1). Therefore, to investigate the effects of these inflammatory cytokines in the BBB after burns, we detected their levels in both serum and brain at different time points after burn trauma in mice. Our findings showed that the levels of IL-6 and IL-1β peaked at 3 h but decreased at 6 and 12 h after burns in serum. Interestingly, the levels of both inflammatory cytokines were increased at 6 h after burn in the brain, which indicated that burn-induced peripheral IL-6 and IL-1β may be the key factors inducing BBB dysfunction and could eventually lead to neuroinflammation. Moreover, these cytokines were found to be neutralized by the injection of UC-MSCs at 1 h after burn trauma. These data supported the hypothesis that the burn might induce an IL-1β- and IL-6-dependent increase in BBB permeability and neuroinflammation in mice and that UC-MSC treatment within 1 h after burn trauma could effectively protect the integrity of BBB and prevent infiltration of inflammatory cytokines including IL-6 and IL-1β.

Western blot analyses showed that the increasing BBB permeability after burn trauma was induced by the decrease in TJs levels and increase in MMP-9 level, which indicated that the burn-induced dysfunction of BBB involved increasing the paracellular pathway. The double bands seen for occludin in the Western blot analyses might be resulted from different structure of the proteins in tissues or the existence of non-specific proteins. Additionally, TEM showed that burns increased the number of vesicles in BBB, which indicated the involvement of transcytosis in increasing the BBB permeability as well. Recent research has confirmed that Mfsd2a deficiency could induce an increase in transcytosis, and compensatory increase in its expression could restore the low permeability of BBB (22, 76). Based on this, our findings suggest that the transcytosis of BBB increased by decreasing the protein level of Mfsd2a after burn trauma. Moreover, UC-MSCs that were injected at 1 h after burn trauma protected TJs, including claudin-5, occludin, and ZO-1, prevented MMP-9 increase, and inhibited transcytosis by enhancing the expression of Mfsd2a, thus recovering the integrity of BBB.

Recent studies illustrated the function of AQP-4 as a water and solute clearance system that is regulated by astrocytes (77). Its normal biological function is to facilitate the removal of waste from the brain (78). Moreover, AQP-4, an intrinsic pro-inflammatory factor, is only activated by the inflammatory factors in the brain tissue and promotes swelling in astrocytes, secretion of inflammatory cytokines (47, 79), as well as the occurrence of cerebral edema (28, 80). In the present study, we detect increased level of AQP-4 in mouse brain after burns, which further confirmed that burns could induce neuroinflammation, mainly via the infiltration of peripheral cytokines. Further, UC-MSCs treatment at 1 h after burns effectively inhibited the increase in AQP-4 level. These results suggest two hypotheses: first, UC-MSCs inhibit the increasing level of AQP-4 by inhibiting the burn-induced neuroinflammation. Second, UC-MSCs directly inhibit the increasing level of AQP-4. However, the underlying mechanism need to be further studied.

Our data collectively indicate that burn trauma induced the disruption of BBB, which is most critical after 6 h. We further identified the potential mechanism by which burns initially increase the BBB permeability: Burns increase the levels of peripheral inflammatory cytokines, including IL-1β and IL-6, which then infiltrate into the CNS by disrupting the BBB and thus induce the neuroinflammation. Li et al. reported that AQP-4, being an intrinsic pro-inflammatory factor, could be activated by the intrinsic neuroinflammation alone (47). Combined with our results, we inferred that the infiltrating cytokines would further activate the expression of AQP-4 and thus exacerbate the neuroinflammation. Our results have already confirmed that increased permeability of BBB after burns was caused by the disruption of TJs, paracellular pathway, decreased Mfsd2a level, and transcytosis. However, the potential mechanism of Mfsd2a decrease after burn trauma needs further investigation. Finally, UC-MSCs inhibited the overexpression of IL-6 and IL-1β, protected the TJs, and impeded the infiltration of pro-inflammatory cytokines into the CNS, thus contributing to the attenuation of CNS neuroinflammation. However, while the potential effects of UC-MSCs on AQP-4 and the underlying mechanisms remain unknown, the decreasing level of AQP-4 in UC-MSCs treated mice further confirmed their efficacy in attenuating the CNS inflammation.

In conclusion, our data suggested that burns could induce an increase in the permeability of BBB via paracellular pathway as well as transcytosis. Specifically, as burns increased the levels of both IL-6 and IL-1β in mice, we inferred that they may affect the permeability of BBB in two ways: First, the increased levels of IL-6 and IL- β observed in the present study induced the disruption of TJs, including claudin-5, occludin and ZO-1, which facilitated the infiltration of peripheral substances into the CNS via paracellular pathway. Second, burns increased the transcytosis in BBB by decreasing the protein level of Mfsd2a, but the underlying mechanism remains to be determined. Moreover, both ways would eventually lead to the CNS inflammation. Subsequently, these blood cytokines would infiltrate into the CNS via either of the two pathways to further induce increase in AQP-4 level, and thus aggravate the neuroinflammation. Finally, we demonstrated that UC-MSCs injected through tail vein in mice at 1 h after burn trauma effectively reversed these adverse effects and thus protect the integrity of BBB. Above all, the present study established a system to study the effects of burn and the injection of UC-MSCs on BBB permeability in mice and investigate the best treatment time point. With fast-paced modern life, the incidences of traumatic injury have increased worldwide, and the complications in CNS induced by peripheral traumatic incidences should be considered. Those who are suffering from such injuries or diseases will be likely afflicted with acute trauma-induced neuroinflammation, which will finally lead to neurological degeneration (81–83). To avoid such eventuality in these cases, it is important to protect the integrity of the key gate—the BBB. Our current research provided a new approach to protect the integrity of BBB from disruption. Administrating UC-MSCs after burn trauma could effectively improve the BBB permeability; thus, we provide evidence as well as basis for translation to the clinic, and further studies are required for evaluating this treatment method.
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: JADICELL -Aegle Therapeutics Announces $4M Financing to Fund Groundbreaking Stem Cell Exosome Clinical Trial

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Aegle Therapeutics Announces $4M Financing to Fund Groundbreaking Stem Cell Exosome Clinical Trial

https://exosome-rna.com/aegle-therapeut ... cal-trial/

Aegle’s capital raise will fund the first clinical trial in the U.S. using exosomes isolated from allogeneic mesenchymal stem cells as therapy.

Aegle Therapeutics Corp., a first in class biotechnology company isolating extracellular vesicles, including exosomes (“EVs”), secreted by mesenchymal stem cells as therapy, today announced the closing of a $4M financing. Aegle’s platform technology is initially being developed to treat dystrophic epidermolysis bullosa (“DEB”), a rare pediatric skin blistering disorder. The investment was led by Boca Raton-based New World Angels, with participation from Tellus BioVentures, DEFTA Healthcare Technologies and DeepWork Capital, as well as exiting investors including OceanAzul Partners, LLC.

Aegle’s technology is based on decades of work conducted by Dr. Evangelos Badiavas, M.D., Ph.D., Aegle’s founder. Aegle’s proprietary isolation process safely and efficiently isolates EVs secreted by allogeneic bone marrow derived mesenchymal stem cells. In preclinical research, Aegle’s EVs demonstrated similar regenerative functionality to their parent cells, opening up the potential for “cell-free” therapy. Additionally, Aegle’s EVs carry specific proteins and mRNA that may prove essential for the treatment of DEB. Aegle’s technology is a platform technology with many potential indications in and beyond dermatology.
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JADICELL -Mesenchymal stromal cells reprogram monocytes and macrophages with processing bodies

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Mesenchymal stromal cells reprogram monocytes and macrophages with processing bodies

https://stemcellsjournals.onlinelibrary ... /stem.3292

Abstract
Mesenchymal stromal cells (MSCs) are widely used in clinical trials because of their ability to modulate inflammation. The success of MSCs has been variable over 25 years, most likely due to an incomplete understanding of their mechanism. After MSCs are injected, they traffic to the lungs and other tissues where they are rapidly cleared. Despite being cleared, MSCs suppress the inflammatory response in the long term. Using human cord tissue‐derived MSCs (hCT‐MSCs), we demonstrated that hCT‐MSCs directly interact and reprogram monocytes and macrophages. After engaging hCT‐MSCs, monocytes and macrophages engulfed cytoplasmic components of live hCT‐MSCs, then downregulated gene programs for antigen presentation and costimulation, and functionally suppressed the activation of helper T cells. We determined that low‐density lipoprotein receptor‐related proteins on monocytes and macrophages mediated the engulfment of hCT‐MSCs. Since a large amount of cellular information can be packaged in cytoplasmic RNA processing bodies (p‐bodies), we generated p‐body deficient hCT‐MSCs and confirmed that they failed to reprogram monocytes and macrophages in vitro and in vivo. hCT‐MSCs suppressed an inflammatory response caused by a nasal lipopolysaccharide challenge. Although both control and p‐body deficient hCT‐MSCs were engulfed by infiltrating lung monocytes and macrophages, p‐body deficient hCT‐MSCs failed to suppress inflammation and downregulate MHC‐II. Overall, we identified a novel mechanism by which hCT‐MSCs indirectly suppressed a T‐cell response by directly interacting and reprogramming monocytes and macrophages via p‐bodies. The results of this study suggest a novel mechanism for how MSCs can reprogram the inflammatory response and have long‐term effects to suppress inflammation.
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JADICELL -Study sheds light on how MSCs suppress inflammation long after they leave the body

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Study sheds light on how MSCs suppress inflammation long after they leave the body

https://www.eurekalert.org/pub_releases ... 110520.php

Durham, NC - A new study released today in STEM CELLS might just have solved the mystery behind why mesenchymal stem cells (MSCs) continue to suppress inflammation in the body long after the MSCs are cleared from the system. The findings, by researchers at Duke University (Durham, N.C.), could help overcome a critical barrier to MSCs being considered a reliable option when developing treatments for inflammatory diseases ranging from COVID-19 and cancer, to allergies, arthritis and more.

MSCs are stem cells that can be isolated from bone marrow, adipose and other tissue sources. Their ability to differentiate into a variety of cell types, along with their capability to self-renew, repair and heal, make them attractive candidates for therapeutic use.

However, there's a drawback, researchers say.

"While MSCs are widely used in clinical trials due to their ability to modulate inflammation, their success over the past 25 years has been varied - something that is likely a result of us not being able to totally understand how they work," said Anthony Filiano, Ph.D. He and Hyunjung Min, Ph.D., in Duke's Marcus Center for Cellular Cures, were co-leaders of the current study.

The ability for MSCs to suppress T cells is well documented in the lab, but little is known how they function in the body. "What we do know," Dr. Filiano continued, "is that after MSCs are injected into the body, they travel to the lungs and other tissues and then are rapidly cleared. Despite this, MSCs suppress the inflammatory response long-term. If the reason behind why and how this is happening can be determined, the information could be very helpful in developing new treatments for a variety of inflammatory diseases."

In search of an answer, the Duke team injected human cord tissue-derived MSCs (hCT-MSCs) into mice with induced lung inflammation. Twenty-four hours later when the mouse lungs were analyzed, "We found that pieces of the MSCs had been engulfed by monocytes and macrophages (types of disease-fighting cells), in effect clearing them from the animals' systems," Dr. Min reported. "We also noted long-term transcription changes in the cells that ate pieces of MSCs suppressed the activation of T cells." (Transcription is the process by which the information in a strand of DNA is copied into a new molecule of messenger RNA.)

Next, using a combination of computational and pharmacological approaches, the researchers identified potential receptors on monocytes and macrophages that mediated their interactions with the hCT-MSCs and blocked interaction with a pharmacological inhibitor. They also identified a key cytoplasmic organelle in hCT-MSCs necessary to reprogram the monocytes and macrophages.

"Our results shed light on how MSCs can modulate the inflammatory response without long-term engraftment using a previously undescribed form of cell communication," said Dr. Filiano, "and explain how MSCs have extended beneficial effects on the body despite being cleared just hours after administration."

Dr. Jan Nolta, Editor-in-Chief of STEM CELLS, said, "this study, which determines a mechanism by which the effects of MSCs on immune modulation are extended after the cells themselves are physically cleared, brings clarity to an longstanding and important question in the field. We congratulate the authors for this important report."
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JADICELL -How One FDA-Approved Stem Cell COVID-19 Treatment Is Being Used in the ICU

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How One FDA-Approved Stem Cell COVID-19 Treatment Is Being Used in the ICU

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JADICELL -True Innovations in Driving Down Cell Therapy Manufacturing

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True Innovations in Driving Down Cell Therapy Manufacturing




What are EXOSOMES

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Re: JADICELL -How I do I feel after getting Stem Cells?

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How I do I feel after getting Stem Cells?

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JADICELL Stem Cell Therapy for Ultrarunners with Dr. Vass and Khoshal | Koopcast Episode 67

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Stem Cell Therapy for Ultrarunners with Dr. Vass and Khoshal | Koopcast Episode 67

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Dr.Santosh Kesari -New Hope on Vanquishing a Relentless Killer | Dr. Santosh Kesari

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New Hope on Vanquishing a Relentless Killer | Dr. Santosh Kesari

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