We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.

Writing Committee for the REMAP-CAP Investigators IMPORTANCE The evidence for benefit of convalescent plasma for critically ill patients with COVID-19 is inconclusive.OBJECTIVE To determine whether convalescent plasma would improve outcomes for critically ill adults with COVID-19. DESIGN, SETTING, AND PARTICIPANTSThe ongoing Randomized, Embedded, Multifactorial, Adaptive Platform Trial for Community-Acquired Pneumonia (REMAP-CAP) enrolled and randomized 4763 adults with suspected or confirmed COVID-19 between March 9, 2020, and January 18, 2021, within at least 1 domain; 2011 critically ill adults were randomized to open-label interventions in the immunoglobulin domain at 129 sites in 4 countries. Follow-up ended on April 19, 2021. INTERVENTIONSThe immunoglobulin domain randomized participants to receive 2 units of high-titer, ABO-compatible convalescent plasma (total volume of 550 mL ± 150 mL) within 48 hours of randomization (n = 1084) or no convalescent plasma (n = 916). MAIN OUTCOMES AND MEASURESThe primary ordinal end point was organ support-free days (days alive and free of intensive care unit-based organ support) up to day 21 (range, −1 to 21 days; patients who died were assigned -1 day). The primary analysis was an adjusted bayesian cumulative logistic model. Superiority was defined as the posterior probability of an odds ratio (OR) greater than 1 (threshold for trial conclusion of superiority >99%). Futility was defined as the posterior probability of an OR less than 1.2 (threshold for trial conclusion of futility >95%). An OR greater than 1 represented improved survival, more organ support-free days, or both. The prespecified secondary outcomes included in-hospital survival; 28-day survival; 90-day survival; respiratory support-free days; cardiovascular support-free days; progression to invasive mechanical ventilation, extracorporeal mechanical oxygenation, or death; intensive care unit length of stay; hospital length of stay; World Health Organization ordinal scale score at day 14; venous thromboembolic events at 90 days; and serious adverse events. RESULTS Among the 2011 participants who were randomized (median age, 61 [IQR, 52 to 70] years and 645/1998 [32.3%] women), 1990 (99%) completed the trial. The convalescent plasma intervention was stopped after the prespecified criterion for futility was met. The median number of organ support-free days was 0 (IQR, -1 to 16) in the convalescent plasma group and 3 (IQR, -1 to 16) in the no convalescent plasma group. The in-hospital mortality rate was 37.3% (401/1075) for the convalescent plasma group and 38.4% (347/904) for the no convalescent plasma group and the median number of days alive and free of organ support was 14 (IQR, 3 to 18) and 14 (IQR, 7 to 18), respectively. The median-adjusted OR was 0.97 (95% credible interval, 0.83 to 1.15) and the posterior probability of futility (OR <1.2) was 99.4% for the convalescent plasma group compared with the no convalescent plasma group. The treatment effects were consistent across the primary outcome and the 11...

Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell-based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donorderived hepatocytes and their pattern of distribution throughout the parenchyma of the recipient's liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n ‫؍‬ 6) or intrahepatic (n ‫؍‬ 6) route into preimmune fetal sheep. The animals were analyzed 56-70 days after transplantation by immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% ؎ 3.5% versus 2.6% ؎ 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. Conclusion: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells. (HEPATOLOGY 2007;46:1935-1945 T he maintenance of cellular homeostasis within the normal liver and during liver regeneration is provided by mature hepatocytes and cholangiocytes and by the intrahepatic (IH) stem cell compartment located in the canals of Hering and the intralobular bile ducts. 1 Recently, another potential source of cells able to provide liver cell replacement has been identified. These stem/progenitor cells consist of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) that reside within the bone marrow (BM) but can reach the liver through the circulatory system. [2][3][4][5][6][7][8][9][10][11][12] Because it is clearly evident that the shortage of available human donor organs cannot meet the needs of all the patients awaiting liver transplantation, alternatives to whole-organ replacement are urgently needed. Cell-based treatments, with cells of either hepatic or extrahepatic origin that would be able to repopulate and re-establish a functional liver after administration, could serve as a possible alternative to wholeorgan transplantation.In order to attain this goal, several questions, such as the sour...

Mesenchymal stem cells (MSCs) possess a set of several fairly unique properties which make them ideally suited both for cellular therapies/regenerative medicine, and as vehicles for gene and drug delivery. These include: 1) relative ease of isolation; 2) the ability to differentiate into a wide variety of seemingly functional cell types of both mesenchymal and non-mesenchymal origin; 3) the ability to be extensively expanded in culture without a loss of differentiative capacity; 4) they are not only hypoimmunogenic, but they produce immunosuppression upon transplantation; 5) their pronounced anti-inflammatory properties; and 6) their ability to home to damaged tissues, tumors, and metastases following in vivo administration. In this review, we summarize the latest research in the use of mesenchymal stem cells in regenerative medicine, as immunomodulatory/anti-inflammatory agents, and as vehicles for transferring both therapeutic genes in genetic disease and genes designed to destroy malignant cells.

Mesenchymal stem cells (MSCs) have been isolated not only from bone marrow, but also from many other tissues such as adipose tissue, skeletal muscle, liver, brain and pancreas. Because MSC were found to have the ability to differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons, hepatocytes and insulin-producing cells, MSCs have generated a great deal of interest for their potential use in regenerative medicine and tissue engineering. Furthermore, given the ease of their isolation and their extensive expansion rate and differentiation potential, mesenchymal stem cells are among the first stem cell types that have a great potential to be introduced in the clinic. Finally, mesenchymal stem cells seem to be not only hypoimmunogenic and thus be suitable for allogeneic transplantation, but they are also able to produce immunosuppression upon transplantation. In this review we summarize the latest research in the use of mesenchymal stem cells in transplantation for generalized diseases, local implantation for local tissue defects, and as a vehicle for genes in gene therapy protocols.

Human bone marrow stromal cells (hBMSCs, also known as bone marrow-derived mesenchymal stem cells) are a population of progenitor cells that contain a subset of skeletal stem cells (hSSCs), able to recreate cartilage, bone, stroma that supports hematopoiesis and marrow adipocytes. As such, they have become an important resource in developing strategies for regenerative medicine and tissue engineering due to their self-renewal and differentiation capabilities. The differentiation of SSCs/BMSCs is dependent on exposure to biophysical and biochemical stimuli that favor early and rapid activation of the in vivo tissue repair process. Exposure to exogenous stimuli such as an electromagnetic field (EMF) can promote differentiation of SSCs/BMSCs via ion dynamics and small signaling molecules. The plasma membrane is often considered to be the main target for EMF signals and most results point to an effect on the rate of ion or ligand binding due to a receptor site acting as a modulator of signaling cascades. Ion fluxes are closely involved in differentiation control as stem cells move and grow in specific directions to form tissues and organs. EMF affects numerous biological functions such as gene expression, cell fate, and cell differentiation, but will only induce these effects within a certain range of low frequencies as well as low amplitudes. EMF has been reported to be effective in the enhancement of osteogenesis and chondrogenesis of hSSCs/BMSCs with no documented negative effects. Studies show specific EMF frequencies enhance hSSC/BMSC adherence, proliferation, differentiation, and viability, all of which play a key role in the use of hSSCs/BMSCs for tissue engineering. While many EMF studies report significant enhancement of the differentiation process, results differ depending on the experimental and environmental conditions. Here we review how specific EMF parameters (frequency, intensity, and time of exposure) significantly regulate hSSC/BMSC differentiation in vitro. We discuss optimal conditions and parameters for effective hSSC/BMSC differentiation using EMF treatment in an in vivo setting, and how these can be translated to clinical trials.

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Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.