Publications by authors named "Darwin J Prockop"

132 Publications

Intranasally Administered Human MSC-Derived Extracellular Vesicles Pervasively Incorporate into Neurons and Microglia in both Intact and Status Epilepticus Injured Forebrain.

Int J Mol Sci 2019 Dec 26;21(1). Epub 2019 Dec 26.

Institute for Regenerative Medicine, Department of Molecular and Cellular Medicine, Texas A&M University College of Medicine, 1114 TAMU, College Station, TX 77842, USA.

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (hMSCs) have great promise as biologics to treat neurological and neurodegenerative conditions due to their robust antiinflammatory and neuroprotective properties. Besides, intranasal (IN) administration of EVs has caught much attention because the procedure is noninvasive, amenable for repetitive dispensation, and leads to a quick penetration of EVs into multiple regions of the forebrain. Nonetheless, it is unknown whether brain injury-induced signals are essential for the entry of IN-administered EVs into different brain regions. Therefore, in this study, we investigated the distribution of IN-administered hMSC-derived EVs into neurons and microglia in the intact and status epilepticus (SE) injured rat forebrain. Ten billion EVs labeled with PKH26 were dispensed unilaterally into the left nostril of naïve rats, and rats that experienced two hours of kainate-induced SE. Six hours later, PKH26 + EVs were quantified from multiple forebrain regions using serial brain sections processed for different neural cell markers and confocal microscopy. Remarkably, EVs were seen bilaterally in virtually all regions of intact and SE-injured forebrain. The percentage of neurons incorporating EVs were comparable for most forebrain regions. However, in animals that underwent SE, a higher percentage of neurons incorporated EVs in the hippocampal CA1 subfield and the entorhinal cortex, the regions that typically display neurodegeneration after SE. In contrast, the incorporation of EVs by microglia was highly comparable in every region of the forebrain measured. Thus, unilateral IN administration of EVs is efficient for delivering EVs bilaterally into neurons and microglia in multiple regions in the intact or injured forebrain. Furthermore, incorporation of EVs by neurons is higher in areas of brain injury, implying that injury-related signals likely play a role in targeting of EVs into neurons, which may be beneficial for EV therapy in various neurodegenerative conditions including traumatic brain injury, stroke, multiple sclerosis, and Alzheimer's disease.
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http://dx.doi.org/10.3390/ijms21010181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981466PMC
December 2019

STAT6 induces expression of Gas6 in macrophages to clear apoptotic neutrophils and resolve inflammation.

Proc Natl Acad Sci U S A 2019 08 30;116(33):16513-16518. Epub 2019 Jul 30.

Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612;

Efferocytosis of apoptotic neutrophils (PMNs) by alveolar macrophages (AMФs) is vital for resolution of inflammation and tissue injury. Here, we investigated the role of AMФ polarization and expression of the efferocytic ligand Gas6 in restoring homeostasis. In the murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), we observed augmented temporal generation of cytokines IL-4 and TSG6 in bronchoalveolar fluid (BALF). Interestingly, we also observed increased expression of antiinflammatory markers consistent with a phenotype shift in AMФs. In particular, AMФs expressed the efferocytic ligand Gas6. In vitro priming of bone marrow-derived macrophages (BMMФs) with IL-4 or TSG6 also induced MФ transition and expression of Gas6. TSG6- or IL-4-primed BMMФs induced efferocytosis of apoptotic PMNs compared with control BMMФs. Adoptive transfer of TSG6- or IL-4-primed BMMФs i.t. into LPS-challenged mice more rapidly and effectively cleared PMNs in lungs compared with control BMMФs. We demonstrated that expression of Gas6 during AMФ transition was due to activation of the transcription factor signal transducer and activator of transcription-6 (STAT6) downstream of IL-4 or TSG6 signaling. Adoptive transfer of Gas6-depleted BMMФs failed to clear PMNs in lungs following LPS challenge and mice showed severely defective resolution of lung injury. Thus, activation of STAT6-mediated Gas6 expression during macrophage phenotype transition resulting in efferocytosis of PMNs plays a crucial role in the resolution of inflammatory lung injury.
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http://dx.doi.org/10.1073/pnas.1821601116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697797PMC
August 2019

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

Curr Eye Res 2019 08 2;44(8):873-881. Epub 2019 Apr 2.

e Department of Surgery, Central Texas Veterans Health Care System , Temple , Texas , United States of America.

: 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. : 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. : The continuous monitoring of corneal alkali injury progression revealed TSG-6 treatments do not show sufficient effectiveness 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. : We conclude that the therapeutic potential of TSG-6 is insufficient in a rat corneal alkali injury model.
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http://dx.doi.org/10.1080/02713683.2019.1597893DOI Listing
August 2019

Macrophage Assay Predicts the Anti-inflammatory Potential of Exosomes from Human Mesenchymal Stromal Cells.

Mol Ther Methods Clin Dev 2019 Jun 15;13:67-76. Epub 2018 Dec 15.

Institute for Regenerative Medicine, Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M University, College Station, TX 77845, USA.

Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed. One important assay will be for the anti-inflammatory activity observed in mice after administration of the small EVs commonly referred to as exosomes. We developed an assay for the anti-inflammatory activity of exosomes with a line of mouse macrophages. The assay makes it possible to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing LPS-stimulated inflammation in mice. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes.
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http://dx.doi.org/10.1016/j.omtm.2018.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350420PMC
June 2019

Human induced pluripotent stem cell-derived MGE cell grafting after status epilepticus attenuates chronic epilepsy and comorbidities via synaptic integration.

Proc Natl Acad Sci U S A 2019 01 17;116(1):287-296. Epub 2018 Dec 17.

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine, Temple, TX 76502;

Medial ganglionic eminence (MGE)-like interneuron precursors derived from human induced pluripotent stem cells (hiPSCs) are ideal for developing patient-specific cell therapy in temporal lobe epilepsy (TLE). However, their efficacy for alleviating spontaneous recurrent seizures (SRS) or cognitive, memory, and mood impairments has never been tested in models of TLE. Through comprehensive video- electroencephalographic recordings and a battery of behavioral tests in a rat model, we demonstrate that grafting of hiPSC-derived MGE-like interneuron precursors into the hippocampus after status epilepticus (SE) greatly restrained SRS and alleviated cognitive, memory, and mood dysfunction in the chronic phase of TLE. Graft-derived cells survived well, extensively migrated into different subfields of the hippocampus, and differentiated into distinct subclasses of inhibitory interneurons expressing various calcium-binding proteins and neuropeptides. Moreover, grafting of hiPSC-MGE cells after SE mediated several neuroprotective and antiepileptogenic effects in the host hippocampus, as evidenced by reductions in host interneuron loss, abnormal neurogenesis, and aberrant mossy fiber sprouting in the dentate gyrus (DG). Furthermore, axons from graft-derived interneurons made synapses on the dendrites of host excitatory neurons in the DG and the CA1 subfield of the hippocampus, implying an excellent graft-host synaptic integration. Remarkably, seizure-suppressing effects of grafts were significantly reduced when the activity of graft-derived interneurons was silenced by a designer drug while using donor hiPSC-MGE cells expressing designer receptors exclusively activated by designer drugs (DREADDs). These results implied the direct involvement of graft-derived interneurons in seizure control likely through enhanced inhibitory synaptic transmission. Collectively, the results support a patient-specific MGE cell grafting approach for treating TLE.
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http://dx.doi.org/10.1073/pnas.1814185115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320542PMC
January 2019

Mesenchymal Stromal Cells Inhibit Inflammatory Lymphangiogenesis in the Cornea by Suppressing Macrophage in a TSG-6-Dependent Manner.

Mol Ther 2018 01 5;26(1):162-172. Epub 2017 Oct 5.

Laboratory of Ocular Regenerative Medicine and Immunology, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea; Department of Ophthalmology, Seoul National University Hospital, Seoul 110-744, Korea. Electronic address:

The cornea is a transparent tissue devoid of blood and lymphatic vessels. However, various inflammatory conditions can cause hemangiogenesis and lymphangiogenesis in the cornea, compromising transparency and visual acuity. Mesenchymal stem/stromal cells (MSCs) have therapeutic potentials in a variety of diseases because of anti-inflammatory properties. Herein, we investigated the effects of MSCs on corneal angiogenesis using a model of suture-induced inflammatory corneal neovascularization. Data demonstrated that an intravenous administration of MSCs suppressed corneal inflammation and neovascularization, inhibiting both hemangiogenesis and lymphangiogenesis. MSCs reduced the levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, Tek, MRC1, and MRC2 in the cornea, which are expressed by pro-angiogenic macrophages. Moreover, the number of CD11b monocytes/macrophages in the cornea, spleen, peripheral blood, and draining lymph nodes was decreased by MSCs. Depletion of circulating CD11b monocytes by blocking antibodies replicated the effects of MSCs. Importantly, knockdown of tumor necrosis factor alpha (TNF-α)-stimulated gene/protein 6 (TSG-6) in MSCs abrogated the effects of MSCs in inhibiting corneal hemangiogenesis and lymphangiogenesis and monocyte/macrophage infiltration. Together, the results suggest that MSCs inhibit inflammatory neovascularization in the cornea by suppressing pro-angiogenic monocyte/macrophage recruitment in a TSG-6-dependent manner.
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http://dx.doi.org/10.1016/j.ymthe.2017.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763076PMC
January 2018

Data against a Common Assumption: Xenogeneic Mouse Models Can Be Used to Assay Suppression of Immunity by Human MSCs.

Mol Ther 2017 08 22;25(8):1748-1756. Epub 2017 Jun 22.

Institute for Regenerative Medicine, College of Medicine, Texas A&M University, 1114 TAMU, 206 Olsen Boulevard, College Station, TX 77845, USA.

Much of what we know about immunology suggests that little is to be gained from experiments in which human cells are administered to immunocompetent mice. Multiple reports have demonstrated that this common assumption does not hold for experiments with human mesenchymal stem/stromal cells (hMSCs). The data demonstrate that hMSCs can suppress immune responses to a variety of stimuli in immunocompetent mice by a range of different mechanisms that are similar to those employed by mouse MSCs. Therefore, further experiments with hMSCs in mice will make it possible to generate preclinical data that will improve both the efficacy and safety of the clinical trials with the cells that are now in progress.
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http://dx.doi.org/10.1016/j.ymthe.2017.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542802PMC
August 2017

MSC-derived Extracellular Vesicles Attenuate Immune Responses in Two Autoimmune Murine Models: Type 1 Diabetes and Uveoretinitis.

Stem Cell Reports 2017 05;8(5):1214-1225

Institute for Regenerative Medicine, College of Medicine, Texas A&M University, 1114 TAMU, 206 Olsen Boulevard, College Station, TX 77845, USA. Electronic address:

Accumulating evidence shows that extracellular vesicles (EVs) produced by mesenchymal stem/stromal cells (MSCs) exert their therapeutic effects in several disease models. We previously demonstrated that MSCs suppress autoimmunity in models of type 1 diabetes (T1D) and experimental autoimmune uveoretinitis (EAU). Therefore, here, we investigated the therapeutic potential of MSC-derived EVs using our established mouse models for autoimmune diseases affecting the pancreas and the eye: T1D and EAU. The data demonstrate that MSC-derived EVs effectively prevent the onset of disease in both T1D and EAU. In addition, the mixed lymphocyte reaction assay with MSC-derived EVs indicated that EVs inhibit activation of antigen-presenting cells and suppress development of T helper 1 (Th1) and Th17 cells. These results raise the possibility that MSC-derived EVs may be an alternative to cell therapy for autoimmune disease prevention.
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http://dx.doi.org/10.1016/j.stemcr.2017.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425726PMC
May 2017

Intranasal MSC-derived A1-exosomes ease inflammation, and prevent abnormal neurogenesis and memory dysfunction after status epilepticus.

Proc Natl Acad Sci U S A 2017 04 10;114(17):E3536-E3545. Epub 2017 Apr 10.

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine, Temple, TX 76502;

Status epilepticus (SE), a medical emergency that is typically terminated through antiepileptic drug treatment, leads to hippocampus dysfunction typified by neurodegeneration, inflammation, altered neurogenesis, as well as cognitive and memory deficits. Here, we examined the effects of intranasal (IN) administration of extracellular vesicles (EVs) secreted from human bone marrow-derived mesenchymal stem cells (MSCs) on SE-induced adverse changes. The EVs used in this study are referred to as A1-exosomes because of their robust antiinflammatory properties. We subjected young mice to pilocarpine-induced SE for 2 h and then administered A1-exosomes or vehicle IN twice over 24 h. The A1-exosomes reached the hippocampus within 6 h of administration, and animals receiving them exhibited diminished loss of glutamatergic and GABAergic neurons and greatly reduced inflammation in the hippocampus. Moreover, the neuroprotective and antiinflammatory effects of A1-exosomes were coupled with long-term preservation of normal hippocampal neurogenesis and cognitive and memory function, in contrast to waned and abnormal neurogenesis, persistent inflammation, and functional deficits in animals receiving vehicle. These results provide evidence that IN administration of A1-exosomes is efficient for minimizing the adverse effects of SE in the hippocampus and preventing SE-induced cognitive and memory impairments.
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http://dx.doi.org/10.1073/pnas.1703920114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410779PMC
April 2017

TNFα-stimulated gene-6 (TSG6) activates macrophage phenotype transition to prevent inflammatory lung injury.

Proc Natl Acad Sci U S A 2016 12 28;113(50):E8151-E8158. Epub 2016 Nov 28.

Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612

TNFα-stimulated gene-6 (TSG6), a 30-kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages are not fully understood. Here we observed markedly augmented LPS-induced inflammatory lung injury and mortality in TSG6 mice compared with WT (TSG6) mice. Treatment of mice with intratracheal instillation of TSG6 prevented LPS-induced lung injury and neutrophil sequestration, and increased survival in mice. We found that TSG6 inhibited the association of TLR4 with MyD88, thereby suppressing NF-κB activation. TSG6 also prevented the expression of proinflammatory proteins (iNOS, IL-6, TNFα, IL-1β, and CXCL1) while increasing the expression of anti-inflammatory proteins (CD206, Chi3l3, IL-4, and IL-10) in macrophages. This shift was associated with suppressed activation of proinflammatory transcription factors STAT1 and STAT3. In addition, we observed that LPS itself up-regulated the expression of TSG6 in TSG6 mice, suggesting an autocrine role for TSG6 in transitioning macrophages. Thus, TSG6 functions by converting macrophages from a proinflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.
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http://dx.doi.org/10.1073/pnas.1614935113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167170PMC
December 2016

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.

Cytotherapy 2017 01 10;19(1):28-35. Epub 2016 Nov 10.

Laboratory of Ocular Regenerative Medicine and Immunology, Seoul National University Hospital, Seoul, Republic of Korea; Department of Ophthalmology, Seoul National University Hospital, Seoul, Republic of Korea. Electronic address:

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)-1β 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.
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http://dx.doi.org/10.1016/j.jcyt.2016.10.007DOI Listing
January 2017

Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs).

Proc Natl Acad Sci U S A 2016 10 3;113(42):E6447-E6456. Epub 2016 Oct 3.

Institute for Regenerative Medicine, College of Medicine, Texas A&M University System Health Science Center, Temple, TX 76502;

Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence.
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http://dx.doi.org/10.1073/pnas.1612290113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081643PMC
October 2016

The exciting prospects of new therapies with mesenchymal stromal cells.

Authors:
Darwin J Prockop

Cytotherapy 2017 01 18;19(1):1-8. Epub 2016 Oct 18.

Institute for Regenerative Medicine, College of Medicine, Texas A&M Health Science Center, Temple, TX, USA. Electronic address:

From the outset, it was apparent that developing new therapies with mesenchymal stem/stromal cells (MSCs) was not a simple or easy task. Among the earliest experiments was administration of MSCs from normal mice to transgenic mice that developed brittle bones because they expressed a mutated gene for type 1 collagen isolated from a patient with osteogenesis imperfecta. The results prompted a clinical trial of MSCs in patients with severe osteogenesis imperfecta. Subsequent work by large numbers of scientists and clinicians has established that, with minor exceptions, MSCs do not engraft or differentiate to a large extent in vivo. Instead the cells produce beneficial effects in a large number of animal models and some clinical trials by secreting paracrine factors and extracellular vesicles in a "hit and run" scenario. The field faces a number of challenges, but the results indicate that we are on the way to effective therapies for millions of patients who suffer from devastating diseases.
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http://dx.doi.org/10.1016/j.jcyt.2016.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154828PMC
January 2017

Stanniocalcin-1 attenuates ischemic cardiac injury and response of differentiating monocytes/macrophages to inflammatory stimuli.

Transl Res 2016 11 9;177:127-142. Epub 2016 Jul 9.

Institute for Regenerative Medicine, Texas A&M University Health Science Center, College of Medicine, Temple, Tex, USA. Electronic address:

Stanniocalcin-1 (STC-1) is a multifunctional glycoprotein with antioxidant and anti-inflammatory properties. Ischemic myocardial necrosis generates "danger" signals that perpetuate detrimental inflammatory reactions often involving monocyte recruitment and their subsequent differentiation into proinflammatory macrophages. Therefore, we evaluated the effects of recombinant STC-1 (rSTC-1) on monocyte phenotype and in a mouse model of myocardial infarction. Using an established protocol to differentiate human monocytes into macrophages, we demonstrated that rSTC-1 did not alter morphology of the differentiated cells, toll-like receptor (TLR) 4 expression, or expression of the myeloid cell marker CD11b. However, rSTC-1 treatment before differentiation attenuated the rise in the expression of CD14, a TLR4 coreceptor and pathogen sensor that propagates innate immune responses, and suppressed levels of inflammatory cytokines produced by the differentiated cells in response to the CD14-TLR4 ligand lipopolysaccharide. Moreover, rSTC-1 treatment reduced CD14 expression in monocytes stimulated with endogenous danger signals. Interestingly, the effects of rSTC-1 on CD14 expression were not reproduced by a superoxide dismutase mimetic. In mice with induced myocardial infarcts, intravenous administration of rSTC-1 decreased CD14 expression in the heart as well as levels of tumor necrosis factor alpha, C-X-C motif ligand 2, interleukin 1 beta, and myeloperoxidase. It also suppressed the formation of scar tissue while enhancing cardiac function. The data suggests that one of the beneficial effects of STC-1 might be attributed to suppression of CD14 on recruited monocytes and macrophages that limits their inflammatory response. STC-1 may be a promising therapy to protect the heart and other tissues from ischemic injury.
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http://dx.doi.org/10.1016/j.trsl.2016.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099094PMC
November 2016

Intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs.

Stem Cell Res Ther 2016 Feb 10;7:27. Epub 2016 Feb 10.

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White, 5701 Airport Rd., Temple, TX, 76502, USA.

Background: Mesenchymal stem/progenitor cells (MSC) have shown beneficial effects in many models of disease in part by modulating excessive inflammatory and immune responses. Frequently the beneficial effects of MSC persist long after their disappearance from host tissues, suggesting that MSC interact with intermediate cells in the host that relay or amplify their effects. The cells have usually been injected intravenously, but beneficial effects have also been reported with intraperitoneal (IP) injection of MSC. However the fate of IP injection of MSC has not been examined.

Methods: The fate of the human MSC injected IP into immune-competent mice was studied. In vivo imaging was used to track green fluorescent protein-labeled MSC in the peritoneal cavity. In addition, their retention in peritoneal tissues was measured by real-time polymerase chain reaction for human GAPDH mRNA. To describe the effects of human MSC on the immune system of the peritoneum, the peritoneal lavage, omentum, lymph nodes and mesenteric tissues were collected. Flow cytometry was used to evaluate the immune cell populations, while cytokine/chemokine production was measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Challenge with lipopolysaccharide at 3 days after the administration of MSC was used to evaluate the preconditioning of the immune system.

Results: Within 20 min, single MSC were no longer detected in peritoneal lavage fluid. Instead they were recovered as aggregates of varying size that contained mouse macrophages and a few B220+ lymphocytes. After 1 day, most of the aggregates containing live MSC were attached to sites throughout the peritoneal cavity including the omentum and mesentery. Less than 0.05 % of the live injected cells were detected in the spleen and jejunal lymph nodes. In all locations, MSC colocalized with mouse macrophages and B220+ lymphocytes. Attachment to the omentum and mesentery was accompanied by the recruitment of immune cells and changes in the production of a series of mouse cytokines. A similar increase in mouse cytokines in the peritoneum was seen after IP injections of human fibroblasts.

Conclusions: IP injected human MSC rapidly formed aggregates with mouse macrophages and B220+ lymphocytes and attached to the walls of the peritoneal cavity. The formation of the aggregates probably limits access of the cells to the systemic circulation.
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http://dx.doi.org/10.1186/s13287-016-0284-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4748482PMC
February 2016

Inflammation, fibrosis, and modulation of the process by mesenchymal stem/stromal cells.

Authors:
Darwin J Prockop

Matrix Biol 2016 04 22;51:7-13. Epub 2016 Jan 22.

Institute for Regenerative Medicine, Texas A&M University, College of Medicine, Temple, TX, USA. Electronic address:

Fibrosis and scarring are the end stage of many disease processes. In effect, the collagen fibers that initially provide a necessary strength during the repair of injured tissues are frequently synthesized in excessive amounts and become irreversible fibrotic deposits that limit regeneration of the endogenous cells of a tissue. This review will focus on the potential of mesenchymal stem/stromal cells for treatment of fibrotic diseases, with emphasis on the role of TSG-6 as a mediator of anti-inflammatory effects.
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http://dx.doi.org/10.1016/j.matbio.2016.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842094PMC
April 2016

Scalable Production of a Multifunctional Protein (TSG-6) That Aggregates with Itself and the CHO Cells That Synthesize It.

PLoS One 2016 21;11(1):e0147553. Epub 2016 Jan 21.

Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.

TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional protein that has a number of potential therapeutic applications. Experiments and clinical trials with TSG-6, however, have been limited by the technical difficulties of producing the recombinant protein. We prepared stable clones of CHO cells that expressed recombinant human TSG-6 (rhTSG-6) as a secreted glycoprotein. Paradoxically, both cell number and protein production decreased dramatically when the clones were expanded. The decreases occurred because the protein aggregated the synthesizing CHO cells by binding to the brush border of hyaluronan that is found around many cultured cells. In addition, the rhTSG-6 readily self-aggregated. To address these problems, we added to the medium an inhibitor of hyaluronan synthesis and heparin to compete with the binding of TSG-6 to hyaluronan. Also, we optimized the composition of the culture medium, and transferred the CHO cells from a spinner culture system to a bioreactor that controlled pH and thereby decreased pH-dependent binding properties of the protein. With these and other improvements in the culture conditions, we obtained 57.0 mg ± 9.16 S.D. of rhTSG-6 in 5 or 6 liter of medium. The rhTSG-6 accounted for 18.0% ± 3.76 S.D. of the total protein in the medium. We then purified the protein with a Ni-chelate column that bound the His tag engineered into the C-terminus of the protein followed by an anion exchange column. The yield of the purified monomeric rhTSG-6 was 4.1 mg to 5.6 mg per liter of culture medium. After intravenous injection into mice, the protein had a longer plasma half-life than commercially available rhTSG-6 isolated from a mammalian cell lysate, apparently because it was recovered as a secreted glycoprotein. The bioactivity of the rhTSG-6 in suppressing inflammation was demonstrated in a murine model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147553PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721919PMC
August 2016

Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI.

Proc Natl Acad Sci U S A 2016 Jan 22;113(1):170-5. Epub 2015 Dec 22.

Institute for Regenerative Medicine at Scott & White, College of Medicine, Texas A&M Health Science Center, Temple, TX 76502

Extracellular vesicles (EVs) secreted by cells present an attractive strategy for developing new therapies, but progress in the field is limited by several issues: The quality of the EVs varies with the type and physiological status of the producer cells; protocols used to isolate the EVs are difficult to scale up; and assays for efficacy are difficult to develop. In the present report, we have addressed these issues by using human mesenchymal stem/stromal cells (MSCs) that produce EVs when incubated in a protein-free medium, preselecting the preparations of MSCs with a biomarker for their potency in modulating inflammation, incubating the cells in a chemically defined protein-free medium that provided a stable environment, isolating the EVs with a scalable chromatographic procedure, and developing an in vivo assay for efficacy of the cells in suppressing neuroinflammation after traumatic brain injury (TBI) in mice. In addition, we demonstrate that i.v. infusion of the isolated EVs shortly after induction of TBI rescued pattern separation and spatial learning impairments 1 mo later.
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http://dx.doi.org/10.1073/pnas.1522297113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711859PMC
January 2016

Mesenchymal stem/stromal cells precondition lung monocytes/macrophages to produce tolerance against allo- and autoimmunity in the eye.

Proc Natl Acad Sci U S A 2016 Jan 22;113(1):158-63. Epub 2015 Dec 22.

Department of Ophthalmology, Seoul National University Hospital, Seoul 110-744, Korea; Laboratory of Ocular Regenerative Medicine and Immunology, Biomedical Research Institute, Seoul National University Hospital, Seoul 110-744, Korea;

Intravenously administered mesenchymal stem/stromal cells (MSCs) engraft only transiently in recipients, but confer long-term therapeutic benefits in patients with immune disorders. This suggests that MSCs induce immune tolerance by long-lasting effects on the recipient immune regulatory system. Here, we demonstrate that i.v. infusion of MSCs preconditioned lung monocytes/macrophages toward an immune regulatory phenotype in a TNF-α-stimulated gene/protein (TSG)-6-dependent manner. As a result, mice were protected against subsequent immune challenge in two models of allo- and autoimmune ocular inflammation: corneal allotransplantation and experimental autoimmune uveitis (EAU). The monocytes/macrophages primed by MSCs expressed high levels of MHC class II, B220, CD11b, and IL-10, and exhibited T-cell-suppressive activities independently of FoxP3(+) regulatory T cells. Adoptive transfer of MSC-induced B220(+)CD11b(+) monocytes/macrophages prevented corneal allograft rejection and EAU. Deletion of monocytes/macrophages abrogated the MSC-induced tolerance. However, MSCs with TSG-6 knockdown did not induce MHC II(+)B220(+)CD11b(+) cells, and failed to attenuate EAU. Therefore, the results demonstrate a mechanism of the MSC-mediated immune modulation through induction of innate immune tolerance that involves monocytes/macrophages.
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http://dx.doi.org/10.1073/pnas.1522905113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711840PMC
January 2016

Hardly Tendentious--Repairing Like with Like.

Authors:
Darwin J Prockop

N Engl J Med 2015 Oct;373(14):1371-2

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http://dx.doi.org/10.1056/NEJMcibr1509841DOI Listing
October 2015

An official American Thoracic Society workshop report: stem cells and cell therapies in lung biology and diseases.

Ann Am Thorac Soc 2015 Apr;12(4):S79-97

The University of Vermont College of Medicine and the Vermont Lung Center, in collaboration with the NHLBI, Alpha-1 Foundation, American Thoracic Society, European Respiratory Society, International Society for Cell Therapy, and the Pulmonary Fibrosis Foundation, convened a workshop, "Stem Cells and Cell Therapies in Lung Biology and Lung Diseases," held July 29 to August 1, 2013 at the University of Vermont. The conference objectives were to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy and ex vivo bioengineering approaches for lung diseases. These are all rapidly expanding areas of study that both provide further insight into and challenge traditional views of mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, discuss and debate current controversies, and identify future research directions and opportunities for both basic and translational research in cell-based therapies for lung diseases. This conference was a follow-up to four previous biennial conferences held at the University of Vermont in 2005, 2007, 2009, and 2011. Each of those conferences, also sponsored by the National Institutes of Health, American Thoracic Society, and Respiratory Disease Foundations, has been important in helping guide research and funding priorities. The major conference recommendations are summarized at the end of the report and highlight both the significant progress and major challenges in these rapidly progressing fields.
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http://dx.doi.org/10.1513/AnnalsATS.201502-086STDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466184PMC
April 2015

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

Proc Natl Acad Sci U S A 2015 Jan 29;112(2):530-5. Epub 2014 Dec 29.

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White, Temple, TX 76502

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 TGFβ, 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.
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http://dx.doi.org/10.1073/pnas.1423008112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299223PMC
January 2015

TSG-6 as a biomarker to predict efficacy of human mesenchymal stem/progenitor cells (hMSCs) in modulating sterile inflammation in vivo.

Proc Natl Acad Sci U S A 2014 Nov 10;111(47):16766-71. Epub 2014 Nov 10.

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott and White, Temple, TX 76502; and

Human mesenchymal stem/progenitor cells (hMSCs) from bone marrow and other tissues are currently being administered to large numbers of patients even though there are no biomarkers that accurately predict their efficacy in vivo. Using a mouse model of chemical injury of the cornea, we found that bone-marrow-derived hMSCs isolated from different donors varied widely in their efficacy in modulating sterile inflammation. Importantly, RT-PCR assays of hMSCs for the inflammation-modulating protein TSG-6 expressed by the TNFα-stimulated gene 6 (TSG-6 or TNFAIP6) predicted their efficacy in sterile inflammation models for corneal injury, sterile peritonitis, and bleomycin-induced lung injury. In contrast, the levels of TSG-6 mRNA were negatively correlated with their potential for osteogenic differentiation in vitro and poorly correlated with other criteria for evaluating hMSCs. Also, a survey of a small cohort suggested that hMSCs from female donors compared with male donors more effectively suppressed sterile inflammation, expressed higher levels of TSG-6, and had slightly less osteogenic potential.
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http://dx.doi.org/10.1073/pnas.1416121111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250139PMC
November 2014

Human adipose-derived stem cells ameliorate cigarette smoke-induced murine myelosuppression via secretion of TSG-6.

Stem Cells 2015 Feb;33(2):468-78

Department of Cellular & Integrative Physiology, Herman B Wells Center for Pediatric Research, Indiana University, Indianapolis, Indiana, USA; Indiana Center for Vascular Biology and Medicine, VC-CAST Signature Center, Department of Medicine, Herman B Wells Center for Pediatric Research, Indiana University, Indianapolis, Indiana, USA; VA Center for Regenerative Medicine Indianapolis, "Richard L. Roudebush" VA Medical Center, Indianapolis, Indiana, USA.

Objective: Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities.

Methods: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony-forming unit assays.

Results: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC.

Conclusion: Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse CS-induced myelosuppression.
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http://dx.doi.org/10.1002/stem.1851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618797PMC
February 2015

Anti-inflammatory protein tumor necrosis factor-α-stimulated protein 6 (TSG-6) promotes early gingival wound healing: an in vivo study.

J Periodontol 2015 Jan;86(1):62-71

Department of Periodontics, Texas A&M University Baylor College of Dentistry, Dallas, TX.

Background: Human multipotent mesenchymal stromal cells (hMSCs) produce tumor necrosis factor (TNF)-α-stimulated protein 6 (TSG-6). TSG-6 modulates proinflammatory cytokine cascades and enhances tissue repair. This study tests the effects of recombinant human TSG-6 (rhTSG-6) on gingival wound healing within the first 2 days post-surgery.

Methods: After gingival resection in 120 Sprague-Dawley rats, 2 µg rhTSG-6 in 5-µL phosphate-buffered saline (PBS) or the same volume of only PBS solution was injected into gingival tissue approximating the surgical wound. Control animals did not receive injections. Tissue biopsies and blood were collected at 1 to 2, 6 to 8, 24, and 48 hours post-surgery (n = 10 per group). Specimens were analyzed via histologic analysis and enzyme-linked immunosorbent assay (ELISA) for quantification and comparison of inflammatory markers interleukin (IL)-1β, IL-6, TNF-α, and myeloperoxidase (MPO). Wound photographs were taken for a double-masked clinical assessment at each time period. Weights were recorded for all animals pre- and post-surgery.

Results: Animals injected with rhTSG-6 had significantly less severe clinical inflammation at 6 to 8 (P = 0.01228), 24 (P = 0.01675), and 48 (P = 0.0186) hours. Sham and control animals had more weight loss at 24 and 48 hours. Sham and control animals had more pronounced cellular infiltrate. rhTSG-6-treated animals had significantly less MPO (P = 0.027) at 24 hours and IL-1β (P = 0.027) at 24 and 48 hours. IL-6 showed a marginal significant difference at 6 to 8 hours, but there was no significant difference for TNF-α.

Conclusion: rhTSG-6 reduced postoperative gingival inflammation by reducing levels of proinflammatory cytokines and cellular infiltrate and may offer significant promise as an anti-inflammatory agent for gingival surgery.
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http://dx.doi.org/10.1902/jop.2014.140187DOI Listing
January 2015

Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions.

Cytotherapy 2014 Nov 16;16(11):1486-1500. Epub 2014 Sep 16.

Institute for Regenerative Medicine, Texas A&M University Health Science Center College of Medicine at Scott & White, Temple, TX, USA. Electronic address:

Background Aims: Human mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations.

Methods: MSC aggregation and sphere formation was studied with the use of hanging drop cultures with medium containing fetal bovine serum or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with the use of gene expression and protein secretion measurements and with functional studies with the use of macrophages and cancer cells.

Results: MSCs did not condense into tight spheroids and express a full complement of therapeutic genes in α-minimum essential medium or several commercial stem-cell media. However, we identified a chemically defined xeno-free media, which, when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (prostaglandin E2, TSG-6 TNF-alpha induced gene/protein 6) and anti-cancer molecules (TRAIL TNF-related apoptosis-inducing ligand, interleukin-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage system and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death.

Conclusions: We demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed in the present study for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications.
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http://dx.doi.org/10.1016/j.jcyt.2014.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190045PMC
November 2014

Rapidly self-renewing human multipotent marrow stromal cells (hMSC) express sialyl Lewis X and actively adhere to arterial endothelium in a chick embryo model system.

PLoS One 2014 21;9(8):e105411. Epub 2014 Aug 21.

Texas A & M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White, Temple, Texas, United States of America.

Background: There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.

Methods: Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.

Results: hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.

Conclusions: The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105411PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140774PMC
May 2015

Are clinical trials with mesenchymal stem/progenitor cells too far ahead of the science? Lessons from experimental hematology.

Stem Cells 2014 Dec;32(12):3055-61

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott and White, Temple, Texas, USA.

The cells referred to as mesenchymal stem/progenitor cells (MSCs) are currently being used to treat thousands of patients with diseases of essentially all the organs and tissues of the body. Strikingly positive results have been reported in some patients, but there have been few prospective controlled studies. Also, the reasons for the beneficial effects are frequently unclear. As a result there has been a heated debate as to whether the clinical trials with these new cell therapies are too far ahead of the science. The debate is not easily resolved, but important insights are provided by the 60-year history that was required to develop the first successful stem cell therapy, the transplantation of hematopoietic stem cells. The history indicates that development of a dramatically new therapy usually requires patience and a constant dialogue between basic scientists and physicians carrying out carefully designed clinical trials. It also suggests that the field can be moved forward by establishing better records of how MSCs are prepared, by establishing a large supply of reference MSCs that can be used to validate assays and compare MSCs prepared in different laboratories, and by continuing efforts to establish in vivo assays for the efficacy of MSCs.
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http://dx.doi.org/10.1002/stem.1806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245369PMC
December 2014

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

Stem Cells Transl Med 2014 May 1;3(5):632-5. Epub 2014 Apr 1.

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine, Temple, Texas, USA.

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.
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http://dx.doi.org/10.5966/sctm.2013-0203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4006492PMC
May 2014

Mesenchymal stromal (stem) cells suppress pro-inflammatory cytokine production but fail to improve survival in experimental staphylococcal toxic shock syndrome.

BMC Immunol 2014 Jan 14;15. Epub 2014 Jan 14.

Sandra A, Rotman Laboratories, Sandra Rotman Centre for Global Health, University Health Network-Toronto General Hospital, University of Toronto, Toronto, M5G 1 L7, Canada.

Background: Toxic shock syndrome (TSS) is caused by an overwhelming host-mediated response to bacterial superantigens produced mainly by Staphylococcus aureus and Streptococcus pyogenes. TSS is characterized by aberrant activation of T cells and excessive release of pro-inflammatory cytokines ultimately resulting in capillary leak, septic shock, multiple organ dysfunction and high mortality rates. No therapeutic or vaccine has been approved by the U.S. Food and Drug Administration for TSS, and novel therapeutic strategies to improve clinical outcome are needed. Mesenchymal stromal (stem) cells (MSCs) are stromal cells capable of self-renewal and differentiation. Moreover, MSCs have immunomodulatory properties, including profound effects on activities of T cells and macrophages in specific contexts. Based on the critical role of host-derived immune mediators in TSS, we hypothesized that MSCs could modulate the host-derived proinflammatory response triggered by Staphylococcal enterotoxin B (SEB) and improve survival in experimental TSS.

Methods: Effects of MSCs on proinflammatory cytokines in peripheral blood were measured in wild-type C57BL/6 mice injected with 50 μg of SEB. Effects of MSCs on survival were monitored in fatal experimental TSS induced by consecutive doses of D-galactosamine (10 mg) and SEB (10 μg) in HLA-DR4 transgenic mice.

Results: Despite significantly decreasing serum levels of IL-2, IL-6 and TNF induced by SEB in wild-type mice, human MSCs failed to improve survival in experimental TSS in HLA-DR4 transgenic mice. Similarly, a previously described downstream mediator of human MSCs, TNF-stimulated gene 6 (TSG-6), did not significantly improve survival in experimental TSS. Furthermore, murine MSCs, whether unstimulated or pre-treated with IFNγ, failed to improve survival in experimental TSS.

Conclusions: Our results suggest that the immunomodulatory effects of MSCs are insufficient to rescue mice from experimental TSS, and that mediators other than IL-2, IL-6 and TNF are likely to play critical mechanistic roles in the pathogenesis of experimental TSS.
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http://dx.doi.org/10.1186/1471-2172-15-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898056PMC
January 2014
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