Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

AbstractThe severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV

Is CD34 truly a negative marker for mesenchymal stromal cells?

The prevailing school of thought is that mesenchymal stromal cells (MSC) do not express CD34, and this sets MSC apart from hematopoietic stem cells (HSC), which do express CD34. However, the evidence for MSC being CD34(-) is largely based on cultured MSC, not tissue-resident MSC, and the existence of CD34(-) HSC is in fact well documented. Furthermore, the Stro-1 antibody, which has been used extensively for the identification/isolation of MSC, was generated by using CD34(+) bone marrow cells as immunogen. Thus, neither MSC being CD34(-) nor HSC being CD34(+) is entirely correct. In particular, two studies that analyzed CD34 expression in uncultured human bone marrow nucleated cells found that MSC (BMSC) existed in the CD34(+) fraction. Several studies have also found that freshly isolated adipose-derived MSC (ADSC) express CD34. In addition, all of these ADSC studies and several other MSC studies have observed a disappearance of CD34 expression when the cells are propagated in culture. Thus the available evidence points to CD34 being expressed in tissue-resident MSC, and its negative finding being a consequence of cell culturing

Effects of EdU labeling on mesenchymal stem cells

BackgroundThymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs.MethodsEdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10(-8) mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay.ResultsEdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express β-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1.DiscussionAt the recommended dosage of 10(-8) mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs

Defining adipose tissue-derived stem cells in tissue and in culture

Adipose tissue-derived stem cells (ADSC) are routinely isolated from the stromal vascular fraction (SVF) of homogenized adipose tissue. Similar to other types of mesenchymal stem cells (MSC), ADSC remain difficult to define due to the lack of definitive cellular markers. Still, many types of MSC, including ADSC, have been shown to reside in a perivascular location, and increasing evidence shows that both MSC and ADSC may in fact be vascular stem cells (VSC). Locally, these cells differentiate into smooth muscle and endothelial cells that are assembled into newly formed blood vessels during angiogenesis and neovasculogenesis. Additionally, MSC or ADSC can also differentiate into tissue cells such as adipocytes in the adipose tissue. Systematically, MSC or ADSC are recruited to injury sites where they participate in the repair/regeneration of the injured tissue. Due to the vasculature’s dynamic capacity for growth and multipotential nature for diversification, VSC in tissue are individually at various stages and on different paths of differentiation. Therefore, when isolated and put in culture, these cells are expected to be heterogeneous in marker expression, renewal capacity, and differentiation potential. Although this heterogeneity of VSC does impose difficulties and cause confusions in basic science studies, its impact on the development of VSC as a therapeutic cell source has not been as apparent, as many preclinical and clinical trials have reported favorable outcomes. With this understanding, ADSC are generally defined as CD34+CD31- although loss of CD34 expression in culture is well documented. In adipose tissue, CD34 is localized to the intima and adventitia of blood vessels but not the media where cells expressing alpha-smooth muscle actin (SMA) exist. By excluding the intima, which contains the CD34+CD31+ endothelial cells, and the media, which contains the CD34-CD31- smooth muscle cells, it leaves the adventitia as the only possible location for the CD34+ ADSC. In the capillary, CD34 and CD140b (a pericyte marker) are mutually exclusively expressed, thus suggesting that pericytes are not the CD34+ ADSC. Many other cellular markers for vascular cells, stem cells, and stem cell niche have also been investigated as possible ADSC markers. Particularly the best-known MSC marker STRO-1 has been found either expressed or not expressed in cultured ADSC. In the adipose tissue, STRO-1 appears to be expressed exclusively in the endothelium of certain but not all blood vessels, and thus not associated with the CD34+ ADSC. In conclusion, we believe that ADSC exist as CD34+CD31-CD104b-SMA- cells in the capillary and in the adventitia of larger vessels. In the capillary these cells coexist with pericytes and endothelial cells, both of which are possibly progenies of ADSC (or more precisely VSC). In the larger vessels, these ADSC or VSC exist as specialized fibroblasts (having stem cell properties) in the adventitia

Effects of EdU labeling on mesenchymal stem cells

BACKGROUND: Thymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been employed for tracking mesenchymal stem cells (MSCs). In the present study we tested whether EdU was cytotoxic and whether it interfered with MSC’s differentiation, cytokine secretion, and migration. METHODS: EdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10 μM of EdU for 48 hours. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression, and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay. RESULTS: EdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine (IBMX) induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express β-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein (LDL) and to form capillary-like structures as well as the expression of vWF, eNOS, and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1. DISCUSSION: At the recommended dosage of 10 μM EdU is non-toxic to ADSCs. EdU label did not interfere with ADSC’s differentiation, cytokine secretion, or migratory response to SDF-1

Recruitment of intracavernously injected adipose-derived stem cells to the major pelvic ganglion improves erectile function in a rat model of cavernous nerve injury

Intracavernous (IC) injection of stem cells has been shown to ameliorate cavernous-nerve (CN) injury-induced erectile dysfunction (ED). However, the mechanisms of action of adipose-derived stem cells (ADSC) remain unclear.status: publishe

Defining Stem and Progenitor Cells within Adipose Tissue

Adipose tissue-derived stem cells (ADSC) are routinely isolated from the stromal vascular fraction (SVF) of homogenized adipose tissue. Freshly isolated ADSC display surface markers that differ from those of cultured ADSC, but both cell preparations are capable of multipotential differentiation. Recent studies have inferred that these progenitors may reside in a perivascular location where they appeared to coexpress CD34 and smooth muscle actin (α-SMA) but not CD31. However, these studies provided only limited histological evidence to support such assertions. In the present study, we employed immunohistochemistry and immunofluorescence to define more precisely the location of ADSC within human adipose tissue. Our results show that α-SMA and CD31 localized within smooth muscle and endothelial cells, respectively, in all blood vessels examined. CD34 localized to both the intima (endothelium) and adventitia neither of which expressed α-SMA. The niche marker Wnt5a was confined exclusively to the vascular wall within mural smooth muscle cells. Surprisingly, the widely accepted mesenchymal stem cell marker STRO-1 was expressed exclusively in the endothelium of capillaries and arterioles but not in the endothelium of arteries. The embryonic stem cell marker SSEA1 localized to a pericytic location in capillaries and in certain smooth muscle cells of arterioles. Cells expressing the embryonic stem cell markers telomerase and OCT4 were rare and observed only in capillaries. Based on these findings and evidence gathered from the existing literature, we propose that ADSC are vascular precursor (stem) cells at various stages of differentiation. In their native tissue, ADSC at early stages of differentiation can differentiate into tissue-specific cells such as adipocytes. Isolated, ADSC can be induced to differentiate into additional cell types such as osteoblasts and chondrocytes