A single intranasal dose of human mesenchymal stem cell-derived extracellular vesicles after traumatic brain injury eases neurogenesis decline, synapse loss, and BDNF-ERK-CREB signaling.

An optimal intranasal (IN) dose of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs), 90 min post-traumatic brain injury (TBI), has been reported to prevent the evolution of acute neuroinflammation into chronic neuroinflammation resulting in the alleviation of long-term cognitive and mood impairments. Since hippocampal neurogenesis decline and synapse loss contribute to TBI-induced long-term cognitive and mood dysfunction, this study investigated whether hMSC-EV treatment after TBI can prevent hippocampal neurogenesis decline and synapse loss in the chronic phase of TBI. C57BL6 mice undergoing unilateral controlled cortical impact injury (CCI) received a single IN administration of different doses of EVs or the vehicle at 90 min post-TBI. Quantifying neurogenesis in the subgranular zone-granule cell layer (SGZ-GCL) through 5'-bromodeoxyuridine and neuron-specific nuclear antigen double labeling at ~2 months post-TBI revealed decreased neurogenesis in TBI mice receiving vehicle. However, in TBI mice receiving EVs (12.8 and 25.6 × 109 EVs), the extent of neurogenesis was matched to naive control levels. A similar trend of decreased neurogenesis was seen when doublecortin-positive newly generated neurons were quantified in the SGZ-GCL at ~3 months post-TBI. The above doses of EVs treatment after TBI also reduced the loss of pre-and post-synaptic marker proteins in the hippocampus and the somatosensory cortex. Moreover, at 48 h post-treatment, brain-derived neurotrophic factor (BDNF), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phosphorylated cyclic AMP response-element binding protein (p-CREB) levels were downregulated in TBI mice receiving the vehicle but were closer to naïve control levels in TBI mice receiving above doses of hMSC-EVs. Notably, improved BDNF concentration observed in TBI mice receiving hMSC-EVs in the acute phase was sustained in the chronic phase of TBI. Thus, a single IN dose of hMSC-EVs at 90 min post-TBI can ease TBI-induced declines in the BDNF-ERK-CREB signaling, hippocampal neurogenesis, and synapses.

Intranasally administered human MSC-derived extracellular vesicles inhibit NLRP3-p38/MAPK signaling after TBI and prevent chronic brain dysfunction.

Traumatic brain injury (TBI) leads to lasting brain dysfunction with chronic neuroinflammation typified by nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome activation in microglia. This study probed whether a single intranasal (IN) administration of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) naturally enriched with activated microglia-modulating miRNAs can avert chronic adverse outcomes of TBI. Small RNA sequencing confirmed the enrichment of miRNAs capable of modulating activated microglia in hMSC-EV cargo. IN administration of hMSC-EVs into adult mice ninety minutes after the induction of a unilateral controlled cortical impact injury resulted in their incorporation into neurons and microglia in both injured and contralateral hemispheres. A single higher dose hMSC-EV treatment also inhibited NLRP3 inflammasome activation after TBI, evidenced by reduced NLRP3, apoptosis-associated speck-like protein containing a CARD, activated caspase-1, interleukin-1 beta, and IL-18 levels in the injured brain. Such inhibition in the acute phase of TBI endured in the chronic phase, which could also be gleaned from diminished NLRP3 inflammasome activation in microglia of TBI mice receiving hMSC-EVs. Proteomic analysis and validation revealed that higher dose hMSC-EV treatment thwarted the chronic activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway by IL-18, which decreased the release of proinflammatory cytokines. Inhibition of the chronic activation of NLRP3-p38/MAPK signaling after TBI also prevented long-term cognitive and mood impairments. Notably, the animals receiving higher doses of hMSC-EVs after TBI displayed better cognitive and mood function in all behavioral tests than animals receiving the vehicle after TBI. A lower dose of hMSC-EV treatment also partially improved cognitive and mood function. Thus, an optimal IN dose of hMSC-EVs naturally enriched with activated microglia-modulating miRNAs can inhibit the chronic activation of NLRP3-p38/MAPK signaling after TBI and prevent lasting brain dysfunction.

Absence of Therapeutic Benefit of the Anti-Inflammatory Protein TSG-6 for Corneal Alkali Injury in a Rat Model.

Purpose: To investigate the therapeutic efficacy of tumor necrosis factor (TNF)-α stimulated gene/protein 6 (TSG-6) in a rat model of corneal alkali injury. Methods: Corneal alkali injury was produced by placing an NaOH-soaked filter paper disk on the central cornea of the right eye of an anesthetized male Lewis (LEW/Crl) rat. Recombinant human TSG-6, or an equal volume of phosphate-buffered saline (PBS), was administered intravenously (IV), by anterior chamber (AC) injection, or as a topical drop. The affected eyes were photographed daily using a dissecting microscope and documented for clinical time course analysis of corneal opacification. Corneal tissue was excised at pre-determined therapeutic endpoints, with subsequent qRT-PCR or histological analyses. Results: The continuous monitoring of corneal alkali injury progression revealed TSG-6 treatments do not show sufficient effectiveness in vivo regardless of IV injection, AC injection, or topical application. Corneal opacification and neovascularization were not diminished, and gene expression was not impacted by these treatments. However, both IV and AC administration of TSG-6 significantly suppressed pro-inflammatory cytokines compared to PBS-treated eyes. Conclusion: We conclude that the therapeutic potential of TSG-6 is insufficient in a rat corneal alkali injury model.

Comparison of the anti-inflammatory effects of induced pluripotent stem cell-derived and bone marrow-derived mesenchymal stromal cells in a murine model of corneal injury.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) offer tremendous potential for therapeutic applications for inflammatory diseases. However, tissue-derived MSCs, such as bone marrow-derived MSCs (BM-MSCs), have considerable donor variations and limited expandability. It was recently demonstrated that MSCs derived from induced pluripotent stem cells (iPSC-MSCs) have less pro-tumor potential and greater expandability of homogenous cell population. In this study, we investigated the anti-inflammatory effects and mechanism of iPSC-MSCs in a murine model of chemical and mechanical injury to the cornea and compared the effects with those of BM-MSCs. METHODS: To create an injury, ethanol was applied to the corneal surface in mice, and the corneal epithelium was removed with a blade. Immediately after injury, mice received an intravenous injection of (i) iPSC-MSCs, (ii) BM-MSCs or (iii) vehicle. Clinical, histological and molecular assays were performed in the cornea to evaluate inflammation. RESULTS: We found that corneal opacity was significantly reduced by iPSC-MSCs or BM-MSCs. Histological examination revealed that the swelling and inflammatory infiltration in the cornea were markedly decreased in mice treated with iPSC-MSCs or BM-MSCs. Corneal levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 were lower in iPSC-MSC- and BM-MSC-treated mice, compared with vehicle-treated controls. In contrast, iPSC-MSCs with a knockdown of the TNF-α stimulating gene (TSG)-6 did not suppress the levels of inflammatory cytokines and failed to reduce corneal opacity. CONCLUSIONS: Together these data demonstrate that iPSC-MSCs exert therapeutic effects in the cornea by reducing inflammation in part through the expression of TSG-6, and the effects are similar to those seen with BM-MSCs.

MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs.

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFß, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

Should publications on mesenchymal stem/progenitor cells include in-process data on the preparation of the cells?

There has been great interest in research and clinical trials with the adult stem/progenitor cells referred to as mesenchymal stem/stromal cells (MSCs). However, there are no definitive markers for the cells and no assays that would reflect the therapeutic efficacy of the cells in vivo. There are in effect no adequate release criteria that define the quality or efficacy of the cells. The problems are compounded by the fact that a variety of different protocols has been used to isolate the cells and expand them in culture. The result is that many publications have used MSCs with different properties, frequently without the investigators being aware of the differences. As a partial solution to these problems, we have devised a simple table to record in-process data on the preparation of MSCs. We suggest that comparisons of data generated by different laboratories would be facilitated if similar in-process data, probably as supplemental materials, were included in publications using MSCs.

Evolving paradigms for repair of tissues by adult stem/progenitor cells (MSCs).

In this review, we focus on the adult stem/progenitor cells that were initially isolated from bone marrow and first referred to as colony forming units-fibroblastic, then as marrow stromal cells and subsequently as either mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). The current interest in MSCs and similar cells from other tissues is reflected in over 10,000 citations in PubMed at the time of this writing with 5 to 10 new publications per day. It is also reflected in over 100 registered clinical trials with MSCs or related cells (http//www.clinicaltrials.gov). As a guide to the vast literature, this review will attempt to summarize many of the publications in terms of three paradigms that have directed much of the work: an initial paradigm that the primary role of the cells was to form niches for haematopoietic stem cells (paradigm I); a second paradigm that the cells repaired tissues by engraftment and differentiation to replace injured cells (paradigm II); and the more recent paradigm that MSCs engage in cross-talk with injured tissues and thereby generate microenvironments or 'quasi-niches' that enhance the repair tissues (paradigm III).

Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses.

Human mesenchymal stromal cells (hMSCs) were injected into the hippocampus of adult mice 1 day after transient global ischemia. The hMSCs both improved neurologic function and markedly decreased neuronal cell death of the hippocampus. Microarray assays indicated that ischemia up-regulated 586 mouse genes. The hMSCs persisted for <7 days, but they down-regulated >10% of the ischemia-induced genes, most of which were involved in inflammatory and immune responses. The hMSCs also up-regulated three mouse genes, including the neuroprotective gene Ym1 that is expressed by activated microglia/macrophages. In addition, the transcriptomes of the hMSC changed with up-regulation of 170 human genes and down-regulation of 54 human genes. Protein assays of the hippocampus demonstrated increased expression in microglia/macrophages of Ym1, the cell survival factor insulin-like growth factor 1, galectin-3, cytokines reflective of a type 2 T cell immune bias, and the major histocompatibility complex II. The observed beneficial effects of hMSCs were largely explained by their modulation of inflammatory and immune responses, apparently by alternative activation of microglia and/or macrophages.

Isolation and culture of bone marrow-derived human multipotent stromal cells (hMSCs).

We have developed protocols whereby a total of 30-90 x 10(6) hMSCs with an average viability greater than 90% can be produced in a single multilevel Cell Factory from a relatively small (1-3 mL) bone marrow aspirate in 14-20 d. It is possible to generate as many as 5 x 10(8) multipotent stromal cells (MSCs) from a single sample, depending on the number of Cell Factories seeded from the initial isolated hMSCs. Briefly, mononuclear cells are collected from a bone marrow aspirate by density gradient centrifugation. The cells are cultured overnight and the adherent cells are allowed to attach to the flask. Nonadherent cells are removed and the culture expanded for 7-10 d with periodic feeding of the cells. The cells are then harvested and seeded at low density (60-100 cells/cm2) into Nunc Cell Factories. The cells are allowed to expand for an additional 7-10 d, and are then harvested.

Differentiation and characterization of human MSCs.

One of the hallmark characteristics of human MSCs (hMSCs) is their ability to differentiate into adipocytes, chondrocytes and osteocytes in culture. The default fate for hMSCs appears to be bone: if late-passage cultures are left in basic culture medium, the hMSCs will become confluent and produce mineral, an indication of bone formation. However, when grown under certain culture conditions or in media containing specific components, the cells can be driven to become a number of other specific cell types including neural cells, myocytes, and cardiomyocytes. The protocols given here are the basic differentiation procedures for inducing osteogenesis, adipogenesis, and chondrogenesis in cultures of hMSCs. Although there is still no clear consensus on the antigen expression pattern that will define hMSCs, a protocol is also presented for the flow cytometric analysis using a series of antibody panels. The analysis of these surface epitope patterns can aide in the isolation and characterization of hMSCs.

Freezing harvested hMSCs and recovery of hMSCs from frozen vials for subsequent expansion, analysis, and experimentation.

Human multipotential stromal cells (hMSCs) are easily isolated from bone marrow and can be expanded by up to 200-fold in culture. Cultures of hMSCs are heterogeneous mixtures of stem/progenitor cells and more mature cell types. The proportion of each cell type in a given culture depends on how the cells are maintained. To maintain their stem cell-like qualities, hMSCs should be plated at low seeding densities (60-150 cells/cm2), lifted when between 60% and 80% confluent and should not be expanded beyond 4-5 passages. Thus, it is useful to establish a frozen bank of early passage cells. hMSCs store well in vapor phase liquid nitrogen (LN2) and are easily recovered for further expansion. This chapter describes one method of establishing a bank of early passage hMSCs using a seed lot system and the subsequent recovery of hMSCs from frozen stocks. The recovered cells can then be harvested and used for analyses of identification, functionality, in vitro and/or in vivo experimentation, or further expanded.

Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice.

We tested the hypothesis that multipotent stromal cells from human bone marrow (hMSCs) can provide a potential therapy for human diabetes mellitus. Severe but nonlethal hyperglycemia was produced in NOD/scid mice with daily low doses of streptozotocin on days 1-4, and hMSCs were delivered via intracardiac infusion on days 10 and 17. The hMSCs lowered blood glucose levels in the diabetic mice on day 32 relative to untreated controls (18.34 mM +/- 1.12 SE vs. 27.78 mM +/- 2.45 SE, P = 0.0019). ELISAs demonstrated that blood levels of mouse insulin were higher in the hMSC-treated as compared with untreated diabetic mice, but human insulin was not detected. PCR assays detected human Alu sequences in DNA in pancreas and kidney on day 17 or 32 but not in other tissues, except heart, into which the cells were infused. In the hMSC-treated diabetic mice, there was an increase in pancreatic islets and beta cells producing mouse insulin. Rare islets contained human cells that colabeled for human insulin or PDX-1. Most of the beta cells in the islets were mouse cells that expressed mouse insulin. In kidneys of hMSC-treated diabetic mice, human cells were found in the glomeruli. There was a decrease in mesangial thickening and a decrease in macrophage infiltration. A few of the human cells appeared to differentiate into glomerular endothelial cells. Therefore, the results raised the possibility that hMSCs may be useful in enhancing insulin secretion and perhaps improving the renal lesions that develop in patients with diabetes mellitus.

Novel object recognition in Apoe(-/-) mice improved by neonatal implantation of wild-type multipotential stromal cells.

Multipotential bone marrow stromal cells (MSCs) from wild-type (Wt) or apolipoprotein E deficient (Apoe(-/-)) mice were implanted into the cerebral ventricles of Apoe(-/-) mice. MSCs from Wt mice continued expressing apoE up to 6 months after implantation and were associated with enhanced novel object recognition and increased microtubule-associated protein 2 (MAP2) immunoreactivity in the dentate gyrus. These data show that MSCs can be used to distinguish developmental from post-developmental effects of a gene knockout and support their therapeutic potential for neurodegenerative diseases.

Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma.

The human adult stem cells from bone marrow stroma referred to as mesenchymal stem cells or marrow stromal cells (MSCs) are of interest because they are easily isolated and expanded and are capable of multipotential differentiation. Here, we examined the ability of recombinant human bone morphogenetic protein (BMP)-2, -4, and -6 to enhance in vitro cartilage formation of MSCs. Human MSCs were isolated from bone marrow taken from normal adult donors. The cells were pelleted and cultured for 21 days in chondrogenic medium containing transforming growth factor beta3 and dexamethasone with or without BMP-2, -4, or -6. All the BMPs tested increased chondrogenic differentiation as assayed by immunohistochemistry and by the size and weight of the cartilage synthesized. However, BMP-2 was the most effective. Microarray analyses of approximately 12,000 genes and reverse transcription-polymerase chain reaction assays established that the critical genes for cartilage synthesis were expressed in the expected time sequence in response to BMP-2. The tissue engineering of autologous cartilage derived from MSCs in vitro for transplantation will be a future alternative for patients with cartilage injuries. To obtain large amounts of cartilage rich in proteoglycans, the use of BMP-2 is recommended, instead of BMP-4 or -6.

Role of PKCalpha in feedback regulation of Na(+) transport in an electrically tight epithelium.

It has long been known that Na(+) channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na(+) channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional (alpha and gamma), novel (delta, eta, and epsilon), and atypical (iota, lambda, and zeta) groups. Steady-state stimulation of Na(+) transport with aldosterone was accompanied by a specific decrease of PKCalpha protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na(+) transport, also decreased PKCalpha levels. These effects were additive, indicating separate mechanisms that converge at the level of PKCalpha. These effects were not accompanied by changes of PKCalpha mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na(+) transport.