Graca Almeida-Porada

Adult mesenchymal stem cells: a pluripotent population with multiple applications.

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.

The effect of low-frequency electromagnetic field on human bone marrow stem/progenitor cell differentiation.

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.

The hematopoietic system in the context of regenerative medicine

Hematopoietic stem cells (HSC) represent the prototype stem cell within the body. Since their discovery, HSC have been the focus of intensive research, and have proven invaluable clinically to restore hematopoiesis following inadvertent radiation exposure and following radio/chemotherapy to eliminate hematologic tumors. While they were originally discovered in the bone marrow, HSC can also be isolated from umbilical cord blood and can be mobilized peripheral blood, making them readily available in relatively large quantities. While their ability to repopulate the entire hematopoietic system would already guarantee HSC a valuable place in regenerative medicine, the finding that hematopoietic chimerism can induce immunological tolerance to solid organs and correct autoimmune diseases has dramatically broadened their clinical utility. The demonstration that these cells, through a variety of mechanisms, can also promote repair/regeneration of non-hematopoietic tissues as diverse as liver, heart, and brain has further increased their clinical value. The goal of this review is to provide the reader with a brief glimpse into the remarkable potential HSC possess, and to highlight their tremendous value as therapeutics in regenerative medicine.

Consensus statement from the first international conference for in utero stem cell transplantation and gene therapy

On April 17–18, 2014, basic and translational scientists and clinicians convened in San Francisco, CA for a conference in fetal stem cell transplantation, stem cell biology, tolerance, and gene therapy. n nThe purpose of the meeting (http://pedsurglab.surgery.ucsf.edu/news–events/fetal-symposium-2014.aspx) was to outline the goals of in utero transplantation, review the barriers that have been encountered, and learn about new developments that can be applied to the field. n nInformation discussed at this conference will help pave the way for developing novel strategies to achieve therapeutic engraftment levels in the fetus, and identify ways to safely translate these strategies to a wide range of clinical applications. n n nWe held a final consensus session to achieve an international agreement for future pre-clinical and clinical studies of in utero hematopoietic cell transplantation (IUHCT). We agreed on the following items: n n nIn utero transplantation is a viable strategy to treat fetuses with selective congenital disorders. n n nGiven recent publications that the maternal immune response can limit engraftment, the clinical strategy for IUHCT should involve transplantation of autologous or maternal-derived cells. The host immune response may be a limiting factor that might be circumvented with early cell delivery. n n nThe fetal microenvironment plays a primary role in supporting the engraftment and expansion of transplanted cells and requires further investigation. n n nRecent data from large animal studies suggests that intravascular injection may be the delivery route of choice to achieve engraftment of hematopoietic stem cells in the fetus. n n nCurrently, there is no proven safe method of host conditioning in the fetus. Until specific, non-toxic conditioning methods (such as antibody-mediated depletion of host HSC) are optimized in pre-clinical models, large cell doses should be used to overcome host competitive barriers. n n nExperimental model data are sufficient to warrant a phase 1 clinical trial of IUHCT for select fetuses. The most suitable hematological diseases are hemoglobinopathies such as sickle cell disease and thalassemia, given their high morbidity/mortality, the availability of reliable prenatal screening programs, and the paucity of optimum postnatal care options. n n nThe value of alternative cells, such as mesenchymal stromal cells (MSC) and amniotic fluid-derived cells, for other appropriate congenital pathologies warrants investigation. n n nReports of using MSC in utero to treat osteogenesis imperfecta (OI) in a limited number of patients are promising and suggest that, after optimization, MSC could be used to improve/treat OI. n n nTreatment of the fetal patient using gene therapy and gene-modified cells have great future potential and should be fields of active investigation. n n nA new society focused on fetal stem cell transplantation and gene therapy will be formed (FeTIS: Fetal Transplantation and Immunology Society), with the mission of accelerating clinical applications of stem cell transplantation and gene therapy approaches to treat fetuses with congenital anomalies. n n nThe society should develop and maintain an international registry of treated patients and their outcomes to facilitate reporting and sharing of results. This database will not be publicly accessible and data will be anonymized. n n nThe society will provide a forum for members to share best practice and clinical governance for in utero stem cell and gene therapy cases.

Transduction of long-term-engrafting human hematopoietic stem cells by retroviral vectors

Gene therapy using retroviral vectors to transfer functional exogenous genes into hematopoietic stem cells (HSCs) promises to provide a permanent cure for a wide array of both hematopoietic and nonhematopoietic disorders by virtue of the fact that retroviral vectors permanently integrate into the host cell genome and HSCs are able to self-renew and give rise to differentiated progeny throughout the life span of the patient. However, for transduction and genomic integration to occur, the target cells must undergo cell division and express the appropriate retroviral receptor, requirements that have thus far hindered attempts at inserting exogenous genes into human HSCs in vitro. In the present studies, we used the fetal sheep xenograft model of human hematopoiesis to evaluate whether human long-term engrafting HSCs could be transduced in vivo, within a fetal microenvironment. We transplanted adult human bone marrow-derived CD34(+)Lin(-) cells into preimmune fetal sheep recipients and subsequently (19 days later) administered clinical-grade murine retroviral vector supernatants to these fetal hematopoietic chimeras. Our results demonstrate that this approach successfully transduced adult human HSCs within all seven sheep that survived the procedure, and that these transduced HSCs had the ability to serially engraft primary, secondary, and tertiary fetal sheep recipients. Transgene expression persisted throughout the serial transplantation. The successful in vivo transduction of long-term engrafting human HSCs with the existing generation of murine retroviral vectors has significant implications for developing new approaches to pre- and postnatal gene therapy.

In utero stem cell transplantation and gene therapy: rationale, history, and recent advances toward clinical application.

Recent advances in high-throughput molecular testing have made it possible to diagnose most genetic disorders relatively early in gestation with minimal risk to the fetus. These advances should soon allow widespread prenatal screening for the majority of human genetic diseases, opening the door to the possibility of treatment/correction prior to birth. In addition to the obvious psychological and financial benefits of curing a disease in utero, and thereby enabling the birth of a healthy infant, there are multiple biological advantages unique to fetal development, which provide compelling rationale for performing potentially curative treatments, such as stem cell transplantation or gene therapy, prior to birth. Herein, we briefly review the fields of in utero transplantation (IUTx) and in utero gene therapy and discuss the biological hurdles that have thus far restricted success of IUTx to patients with immunodeficiencies. We then highlight several recent experimental breakthroughs in immunology, hematopoietic/marrow ontogeny, and in utero cell delivery, which have collectively provided means of overcoming these barriers, thus setting the stage for clinical application of these highly promising therapies in the near future.

Indocyanine green loaded hyaluronan-derived nanoparticles for fluorescence-enhanced surgical imaging of pancreatic cancer

Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. In this study, hyaluronic acid derived nanoparticles with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG was not cytotoxic to healthy pancreatic epithelial cells and did not induce chemotaxis or phagocytosis, it accumulated significantly within the pancreas in an orthotopic pancreatic ductal adenocarcinoma model, and demonstrated contrast-enhancement for pancreatic lesions relative to non-diseased portions of the pancreas. Fluorescence microscopy showed higher fluorescence intensity in pancreatic lesions and splenic metastases due to NanoICG compared to ICG alone. The in vivo safety profile of NanoICG, including, biochemical, hematological, and pathological analysis of NanoICG-treated healthy mice, indicates negligible toxicity. These results suggest that NanoICG is a promising contrast agent for intraoperative detection of pancreatic tumors.

In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells

Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation, and likely one or more solar particle events (SPEs). Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions. We show that exposing human hematopoietic stem/progenitor cells (HSC) to extended mission-relevant doses of accelerated high-energy protons and iron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces in vitro colony formation; (3) markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, of what appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone. Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.

Boosting Hematopoietic Engraftment after in Utero Transplantation through Vascular Niche Manipulation

Summary In utero hematopoietic stem/progenitor cell transplantation (IUHSCT) has only been fully successful in the treatment of congenital immunodeficiency diseases. Using sheep as a large animal model of IUHSCT, we demonstrate that administration of CD146+CXCL12+VEGFR2+ or CD146+CXCL12+VEGFR2− cells prior to, or in combination with, hematopoietic stem/progenitor cells (HSC), results in robust CXCL12 production within the fetal marrow environment, and significantly increases the levels of hematopoietic engraftment. While in the fetal recipient, donor-derived HSC were found to reside within the trabecular bone, the increased expression of VEGFR2 in the microvasculature of CD146+CXCL12+VEGFR2+ transplanted animals enhanced levels of donor-derived hematopoietic cells in circulation. These studies provide important insights into IUHSCT biology, and demonstrate the feasibility of enhancing HSC engraftment to levels that would likely be therapeutic in many candidate diseases for IUHSCT.

Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models

Selectively recruiting bone marrow (BM)‐derived stem and progenitor cells to injury sites is a promising therapeutic approach. The coordinated action of soluble factors is thought to trigger the mobilization of stem cells from the BM and recruit them to lesions to contribute to tissue regeneration. Nevertheless, the temporal response profile of the major cellular players and soluble factors involved in priming the BM and recruiting BM‐derived cells to promote regeneration is unknown. We show that injury alters the BM cellular composition, introducing population‐specific fluctuations during tissue regeneration. We demonstrate that injury causes an immediate, transient response of mesenchymal stromal cells and endothelial cells followed by a nonoverlapping increase in hematopoietic stem and progenitor cells. Moreover, BM reaction is identical whether the injury is inflicted on skin and muscle or also involves a bone defect, but these 2 injury paradigms trigger distinct systemic cytokine responses. Together, our results indicate that the BM response to injury in the early stages of regeneration is independent of the tissue‐of‐injury based on the 2 models used, but the injured tissue dictates the systemic cytokine response.—Leitão, L., Alves, C. J., Alencastre, I. S., Sousa, D. M., Neto, E., Conceição, F., Leitão, C., Aguiar, P., Almeida‐Porada, G., Lamghari, M. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue‐of‐injury in 2 injury models. FASEB J. 33, 857–872 (2019). www.fasebj.org