A Novel Method for the Production of an Autologous Anti-Inflammatory and Anti-Catabolic Product (Cytorich) from Human Blood: A Prospective Treatment for the COVID-19-Induced Cytokine Storm.

BACKGROUND Autologous blood-derived products can target specific inflammatory molecular pathways and have potentially beneficial therapeutic effects on inflammatory pathologies. The purpose of this study was to assess in vitro the anti-inflammatory and anti-catabolic potential of an autologous blood product as a possible treatment for COVID-19-induced cytokine storm. MATERIAL AND METHODS Blood samples from healthy donors and donors who had recovered from COVID-19 were incubated using different techniques and analyzed for the presence of anti-inflammatory, anti-catabolic, regenerative, pro-inflammatory, and procatabolic molecules. RESULTS The highest concentrations of therapeutic molecules for targeting inflammatory pathways were found in the blood that had been incubated for 24 h at 37°C, whereas a significant increase was observed after 6 h of incubation in blood from COVID-19-recovered donors. Beneficially, the 6-h incubation process did not downregulate anti-COVID-19 immunoglobulin G concentrations. Unfortunately, increases in matrix metalloproteinase 9, tumor necrosis factor alpha, and interleukin-1 were detected in the product after incubation; however, these increases could be blocked by adding citric acid, with no effect on the concentration of the target therapeutic molecules. Our data allow for safer and more effective future treatments. CONCLUSIONS An autologous blood-derived product containing anti-inflammatory and anti-catabolic molecules, which we term Cytorich, has a promising therapeutic role in the treatment of a virus-induced cytokine storm, including that associated with COVID-19.

Dual embryonic origin of the mammalian enteric nervous system.

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.

The neural stem cell lineage reveals novel relationships among spermatogonial germ stem cells and other pluripotent stem cells.

The embryonic stem cell (ESC) derived from the inner cell mass is viewed as the core pluripotent cell (PC) type from which all other cell types emanate. This familiar perspective derives from an embryological time line in which PCs are ordered according to their time of appearance. However, this schema does not take into account their potential for interconversion, thereby excluding this critical quality of PCs. The persistence of bona fide pluripotent adult stem cells has garnered increasing attention in recent years. Adult pluripotent spermatogonial germ stem cells (aSGSCs) arise from primordial germ cells (pGCs) that emerge from the epiblast during gastrulation. Adult definitive neural stem cells (dNSCs) arise clonally from pluripotent embryonic primitive neural stem cells (pNSCs), which can also be derived clonally from ESCs. To test for stem cell-type convertibility, we employed differentiation in the clonal lineage from ESCs to pNSCs to dNSCs, and revealed the relationships and lineage positioning among various PC populations, including spermatogonial germ cells (aSGSCs), epiblast-derived stem cells (Epi-SCs) and the bFGF, Activin, and BIO-derived stem cell (FAB-SC). Adult, murine aSGSCs assumed a 'pseudo-ESC' state in vitro, and then differentiated into dNSCs, but not pNSCs. Similarly, Epi-SCs and FAB-SCs only gave rise to dNSCs and not to pNSCs. The results of these experiments suggest a new pluripotency lineage model describing the relationship(s) among PCs that better reflects the transitions between these cell types in vitro.

Oct4 is required ~E7.5 for proliferation in the primitive streak.

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

Generation of neural crest cells and peripheral sensory neurons from human embryonic stem cells.

Peripheral somatic sensory neurons (PSNs) are responsible for the critical function of transmitting multiple modalities of information from the outside world, including heat, touch, and pain, as well as the position of muscles required for coordinated voluntary movement to the central nervous system. Many peripheral neuropathies exist, including hereditary neurodegeneration in Familial Dysautonomia, infections of PSNs by viruses such as Varicella zoster and damage to PSNs and/or their process resulting from other disease conditions such as diabetes. Understanding of the etiology of these diseases and development of treatments is hampered by the lack of normal and healthy human PSNs for study, which are only available from abortuses or rare surgical procedures.Human embryonic stem cells (hESCs) are an ideal source of cells for generating normal PSNs for study of disease and drug development, since they can be grown virtually indefinitely in tissue culture and have the potential to form any cell type in the body. Several years ago, we generated human neurons with the molecular characteristics of PSNs from hESCs at low (less than 1%) yields (Pomp et al., Stem Cells 23:923-930, 2005). The present chapter details our most recently improved method that uses 2 rounds of PA6-induction to rapidly generate PSNs at more than 25% purity (Pomp et al., Br. Res. 1230: 50-60, 2008).The neural crest (NC) is a transient multipotent embryonic stem cell population that is the source of PSNs. NC cells give rise to diverse and important tissues in man, but human NC has not been studied because of the difficulty in obtaining 3-5 week human embryos. The methods described in this chapter can also be used to quickly generate large numbers of human NC for study.

Genetic modification of human embryonic stem cells with adenoviral vectors: differences of infectability between lines and correlation of infectability with expression of the coxsackie and adenovirus receptor.

Adenovirus is an efficient vector for expression of transgenes in dividing and nondividing cells. However, very few studies of human embryonic stem cells (hESCs) have utilized adenoviral vectors. We examine here the ability of adenovirus to infect naive hESCs and the differentiated derivatives of multiple hESC lines. We found a striking variation in adenovirus infection rates between lines. The variability in infection rates was positively correlated with the expression of the coxsackievirus and adenovirus receptor, but not that of alpha(nu)-integrin. Adenoviral infection did not interfere with the expression of pluripotency markers, even after passaging. In addition, infection did not affect differentiation of hESC-derived neural precursors in vitro. We also found that green fluorescent protein expression mediated by adenovirus can be a useful marker for tracking hESC in xenografts. We conclude that adenovirus is a practical vector for genetic modification of naive hESC from most, but not all lines, but may be more generally useful for gene transfer into differentiated derivatives of hESC lines.

PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells.

Human embryonic stem cells (hESC) have been directed to differentiate into CNS cells with clinical importance. However, for study of development and regeneration of the human PNS, and peripheral neuropathies, it would be useful to have a source of human PNS derivatives. We have demonstrated that peripheral sensory neuron-like cells (PSN) can also be derived from hESC via neural crest-like (NC) intermediates, and from neural progenitors induced from hESC using noggin. Here we report the generation of higher purity PSN from passagable neurospheres (NSP) induced by murine PA6 stromal cells. hESC were cultured with PA6, and colonies that developed a specific morphology were cut from the plates. Culture of these colonies under non-adhesive conditions yielded NSPs. Several NC marker genes were expressed in the NSP, and these were also detected in 3-5week gestation human embryos containing migrating NC. These NSPs passaged for 2-8weeks and re-plated on PA6 gave rise to many Brn3a+/peripherin+ cells, characteristic of early sensory-like neurons. Re-culturing PA6-induced NSP cells with PA6 resulted in about 25% of the human cells in the co-cultures differentiating to PSN after 1week, compared to only about 10% PSN obtained after 3 weeks when noggin-induced NSP were used. Two month adherent cultures of PA6-induced NSP cells contained neurons expressing several PSN neuropeptides, and voltage-dependent currents and action potentials were obtained from a molecularly identified PSN. hESC-derived PA6-induced NSP cells are therefore an excellent potential source of human PSN for study of differentiation and modeling of PNS disease.

Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells.

Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naive hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.

Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells.

Human embryonic stem cells (hESCs) have been directed to differentiate into neuronal cells using many cell-culture techniques. Central nervous system cells with clinical importance have been produced from hESCs. To date, however, there have been no definitive reports of generation of peripheral neurons from hESCs. We used a modification of the method of Sasai and colleagues for mouse and primate embryonic stem cells to elicit neuronal differentiation from hESCs. When hESCs are cocultured with the mouse stromal line PA6 for 3 weeks, neurons are induced that coexpress (a) peripherin and Brn3a, and (b) peripherin and tyrosine hydroxylase, combinations characteristic of peripheral sensory and sympathetic neurons, respectively. In vivo, peripheral sensory and sympathetic neurons develop from the neural crest (NC). Analysis of expression of mRNAs identified in other species as NC markers reveals that the PA6 cells induce NC-like cells before neuronal differentiation takes place. Several NC markers, including SNAIL, dHAND, and Sox9, are increased at 1 week of coculture relative to naive cells. Furthermore, the expression of several NC marker genes known to be downregulated upon in vivo differentiation of NC derivatives, was observed to be present at lower levels at 3 weeks of PA6-hESC coculture than at 1 week. Our report is the first on the expression of molecular markers of NC-like cells in primates, in general, and in humans, specifically. Our results suggest that this system can be used for studying molecular and cellular events in the almost inaccessible human NC, as well as for producing normal human peripheral neurons for developing therapies for diseases such as familial dysautonomia.