Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy.

BACKGROUND AIMS: To obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) must be expanded ex vivo. METHODS: A retrospective analysis was performed of records of 76 autologous MSC products used in phase I or II clinical studies performed in a cohort of cardiovascular patients. In all cases, native MSCs present in patient bone marrow aspirates were separated and expanded ex vivo. RESULTS: The cell products were classified in two groups (A and B), according to biologic properties and expansion time (ex vivo passages) to reach the protocol-established cell dose. In group A, the population of adherent cells obtained during the expansion period (2 ± 1 passages) was composed entirely of MSCs and met the requirements of cell number and biologic features as established in the respective clinical protocol. In group B, in addition to MSCs, we observed during expansion a high proportion of ancillary cells, characterized as osteoclast precursor cells. In this case, although the biologic properties of the resulting MSC product were not affected, the yield of MSCs was significantly lower. The expansion cycles had to be increased (3 ± 1 passages). CONCLUSIONS: These results suggest that the presence of osteoclast precursor cells in bone marrow aspirates may impose a limit for the proper clinical use of ex vivo expanded autologous bone marrow-derived MSCs.

Vascular disease and stem cell therapies.

INTRODUCTION AND BACKGROUND: Peripheral vascular disease is the leading cause of limb ischemia (LI). LI is manifested by claudication, ischemic rest pain, ulcers or gangrene. It is the result of peripheral arterial disease due to atherosclerosis. Over the last decade, several centers around the world have initiated clinical trials utilizing stem cells as a treatment for this disease. SOURCES OF DATA: Published medical literature, clinical trials announced in clinical trials.gov and TCA cellular therapy experience. AREAS OF AGREEMENT: There is general agreement that stem cells are useful for LI. AREAS OF CONTROVERSY: These arise from the type of cells, dose, route of administration and methods to evaluate efficacy. GROWING POINTS: Growing evidence suggests that bone marrow derived-mesenchymal stem cells are as good as or superior to mononuclear cells, and a combination of both cell types may be even better. AREAS TIMELY FOR DEVELOPING RESEARCH: Based on current trials and publications, several promising biological products could become part of the therapeutic arsenal for LI. This may include combinations of more than one type of adult/induced pluripotent stem cells/embryonic stem cells, use of stem cells with growth factors or extracellular matrix molecules.

Bone marrow-derived stem/progenitor cells: their use in clinical studies for the treatment of myocardial infarction.

Over the last six years, several centres around the world have started clinical trials to investigate the utilisation of bone marrow-derived cells for myocardial infarction. Different types and numbers of cells have been used assuming they possess a potential to originate new endothelial cells and/or cardiomyocytes to repair/regenerate the ailed heart. Despite diversity in number, clinical status of subjects, route of cell administration, and criteria to evaluate efficacy, the main conclusion drawn from these clinical studies was that such therapies were safe. However, attempts to unify efficacy data have yielded no clear answers, so far. This review offers an in-depth and critical analysis of these trials and intends to evaluate from the cellular biology and clinical cardiology viewpoints, the significant information that has been published since 2002, as well as that emerging from ongoing clinical trials. Emphasis will be placed on cellular types, research designs and methods to evaluate efficacy of each particular treatment modality.

Treatment of osteonecrosis of the femoral head by core decompression and implantation of fully functional ex vivo-expanded bone marrow-derived mesenchymal stem cells: a proof-of-concept study.

BACKGROUND: Based on several attributes involved in bone formation, bone marrow-resident mesenchymal stem cells (MSCs) have been employed in the treatment of patients suffering from femoral head osteonecrosis. Due to the low content of MSCs in the bone marrow, ex vivo expansion procedures are utilized to increase the cell number. Customarily, before administration of the resulting expanded cell product MSCs to the patient, its cellular identity is usually evaluated according to a set of "minimal phenotypic" markers, which are not modified by ex vivo processing. However, MSC functional ("reparative") markers, which are severely impaired along the ex vivo expansion routine, are usually not assessed. PATIENTS AND METHODS: In this proof-of-concept study, a cohort of five avascular osteonecrosis patients received an instillation of ex vivo-expanded autologous MSCs, manufactured under controlled conditions, with an aim to protect their functional ("reparative") capacity. RESULTS AND CONCLUSION: Outcomes of this study confirmed the safety and effectiveness of the MSC-based therapy used. After a follow-up period (19-54 months), in all patients, the hip function was significantly improved and pain intensity markedly reduced. As a corollary, no patient required hip arthroplasty.

Comparative study of the neural differentiation capacity of mesenchymal stromal cells from different tissue sources: An approach for their use in neural regeneration therapies.

Mesenchymal stem cells (MSCs) can trans/differentiate to neural precursors and/or mature neurons and promote neuroprotection and neurogenesis. The above could greatly benefit neurodegenerative disorders as well as in the treatment of post-traumatic and hereditary diseases of the central nervous system (CNS). In order to attain an ideal source of adult MSCs for the treatment of CNS diseases, adipose tissue, bone marrow, skin and umbilical cord derived MSCs were isolated and studied to explore differences with regard to neural differentiation capacity. In this study, we demonstrated that MSCs from several tissues can differentiate into neuron-like cells and differentially express progenitors and mature neural markers. Adipose tissue MSCs exhibited significantly higher expression of neural markers and had a faster proliferation rate. Our results suggest that adipose tissue MSCs are the best candidates for the use in neurological diseases.

Mesenchymal stem cell therapy in the treatment of hip osteoarthritis.

This study was performed to investigate the safety and efficacy of the intra-articular infusion of ex vivo expanded autologous bone marrow-derived mesenchymal stem cells (BM-MSC) to a cohort of patients with articular cartilage defects in the hip. The above rationale is sustained by the notion that MSCs express a chondrocyte differential potential and produce extracellular matrix molecules as well as regulatory signals, that may well contribute to cure the function of the damaged hip joint. A cohort of 10 patients with functional and radiological evidences of hip osteoarthritis, either in one or both legs, was included in the study. BM-MSC (the cell product) were prepared and infused into the damaged articulation(s) of each patient (60 × 106 cells in 3 weekly/doses). Before and after completion of the cell infusion scheme, patients were evaluated (hip scores for pain, stiffness, physical function, range of motion), to assess whether the infusion of the respective cell product was beneficial. The intra-articular injection of three consecutive weekly doses of ex vivo expanded autologous BM-MSC to patients with articular cartilage defects in the hip and proved to be a safe and clinically effective treatment in the restoration of hip function and range of motion. In addition, the statistical significance of the above data is in line with the observation that the radiographic scores (Tönnis Classification of Osteoarthritis) of the damaged leg(s) remained without variation in 9 out of 10 patients, after the administration of the cell product.

Adult hippocampus derived soluble factors induce a neuronal-like phenotype in mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) are not restricted in their differentiation fate to cells of the mesenchymal lineage. They acquire a neural phenotype in vitro and in vivo after transplantation in the central nervous system. Here we investigated whether soluble factors derived from different brain regions are sufficient to induce a neuronal phenotype in MSCs. We incubated bone marrow-derived MSCs in conditioned medium (CM) derived from adult hippocampus (HCM), cortex (CoCM) or cerebellum (CeCM) and analyzed the cellular morphology and the expression of neuronal and glial markers. In contrast to muscle derived conditioned medium, which served as control, conditioned medium derived from the different brain regions induced a neuronal morphology and the expression of the neuronal markers GAP-43 and neurofilaments in MSCs. Hippocampus derived conditioned medium had the strongest activity. It was independent of NGF or BDNF; and it was restricted to the neuronal differentiation fate, since no induction of the astroglial marker GFAP was observed. The work indicates that soluble factors present in the brain are sufficient to induce a neuronal phenotype in MSCs.

Mesenchymal stem cells and the treatment of cardiac disease.

The ischemia-induced death of cardiomyocytes results in scar formation and reduced contractility of the ventricle. Several preclinical and clinical studies have supported the notion that cell therapy may be used for cardiac regeneration. Most attempts for cardiomyoplasty have considered the bone marrow as the source of the "repair stem cell(s)," assuming that the hematopoietic stem cell can do the work. However, bone marrow is also the residence of other progenitor cells, including mesenchymal stem cells (MSCs). Since 1995 it has been known that under in vitro conditions, MSCs differentiate into cells exhibiting features of cardiomyocytes. This pioneer work was followed by many preclinical studies that revealed that ex vivo expanded, bone marrow-derived MSCs may represent another option for cardiac regeneration. In this work, we review evidence and new prospects that support the use of MSCs in cardiomyoplasty.

Nonstimulated human uncommitted mesenchymal stem cells express cell markers of mesenchymal and neural lineages.

Ex vivo cultures of human bone marrow-derived mesenchymal stem cells (MSCs) contain subsets of progenitors exhibiting dissimilar properties. One of these subsets comprises uncommitted progenitors displaying distinctive features, such as morphology, a quiescent condition, growth factor production, and restricted tissue biodistribution after transplantation. In this study, we assessed the competence of these cells to express, in the absence of differentiation stimuli, markers of mesoderm and ectodermic (neural) cell lineages. Fluorescence microscopy analysis showed a unique pattern of expression of osteogenic, chondrogenic, muscle, and neural markers. The depicted "molecular signature" of these early uncommitted progenitors, in the absence of differentiation stimuli, is consistent with their multipotentiality and plasticity as suggested by several in vitro and in vivo studies.

Cell Therapy and Tissue Engineering Approaches for Cartilage Repair and/or Regeneration.

Articular cartilage injuries caused by traumatic, mechanical and/or by progressive degeneration result in pain, swelling, subsequent loss of joint function and finally osteoarthritis. Due to the peculiar structure of the tissue (no blood supply), chondrocytes, the unique cellular phenotype in cartilage, receive their nutrition through diffusion from the synovial fluid and this limits their intrinsic capacity for healing. The first cellular avenue explored for cartilage repair involved the in situ transplantation of isolated chondrocytes. Latterly, an improved alternative for the above reparative strategy involved the infusion of mesenchymal stem cells (MSC), which in addition to a self-renewal capacity exhibit a differentiation potential to chondrocytes, as well as a capability to produce a vast array of growth factors, cytokines and extracellular matrix compounds involved in cartilage development. In addition to the above and foremost reparative options up till now in use, other therapeutic options have been developed, comprising the design of biomaterial substrates (scaffolds) capable of sustaining MSC attachment, proliferation and differentiation. The implantation of these engineered platforms, closely to the site of cartilage damage, may well facilitate the initiation of an 'in situ' cartilage reparation process. In this mini-review, we examined the timely and conceptual development of several cell-based methods, designed to repair/regenerate a damaged cartilage. In addition to the above described cartilage reparative options, other therapeutic alternatives still in progress are portrayed.

Marrow-derived mesenchymal stem cells: role in epithelial tumor cell determination.

Marrow stroma represents an advantageous environment for development of micrometastatic cells. Within the cellular structure of marrow stroma, mesenchymal stem cells (MSC) have been postulated as an interacting target for disseminated cancer cells. The studies reported here were performed to gain more information on the interaction of the human breast cancer cell line MCF-7 with human bone marrow-derived MSC cells and to investigate whether this interaction affects tumor cell properties. The results showed that after co-culture with MSC, changes were detected in the morphology, proliferative capacity and aggregation pattern of MCF-7 cells, but these parameters were not affected after the co-culture of MSC cells with a non-tumorigenic breast epithelial cell line, MCF-10. Since the indirect culture of MCF-7 with MSC or its products also resulted in functional changes in the tumor cells, we evaluated whether these effects could be attributed to growth factors produced by MSC cells. It was found that VEGF and IL-6 mimic the effects produced by MSC or its products on the proliferation and aggregation properties of MCF-7, cells, respectively. Thus, it seems that after entry of disseminated tumor cells into the marrow space, their proliferative and morphogenetic organization patterns are modified after interaction with distinct stromal cells and/or with specific signals from the marrow microenvironment.

Dynamic of distribution of human bone marrow-derived mesenchymal stem cells after transplantation into adult unconditioned mice.

BACKGROUND: The use of mesenchymal stem cells (MSC) for cell therapy relies on their capacity to engraft and survive long-term in the appropriate target tissue(s). Animal models have demonstrated that the syngeneic or xenogeneic transplantation of MSC results in donor engraftment into the bone marrow and other tissues of conditioned recipients. However, there are no reliable data showing the fate of human MSC infused into conditioned or unconditioned adult recipients. METHODS: In the present study, the authors investigated, by using imaging, polymerase chain reaction (PCR), and in situ hybridization, the biodistribution of human bone marrow-derived MSC after intravenous infusion into unconditioned adult nude mice. RESULTS: As assessed by imaging (gamma camera), PCR, and in situ hybridization analysis, the authors' results demonstrate the presence of human MSC in bone marrow, spleen, and mesenchymal tissues of recipient mice. CONCLUSIONS: These results suggest that human MSC transplantation into unconditioned recipients represents an option for providing cellular therapy and avoids the complications associated with drugs or radiation conditioning.

Human cord blood-derived mesenchymal stem cells home and survive in the marrow of immunodeficient mice after systemic infusion.

Bone marrow is the residence site of mesenchymal stem cells (MSC), which upon commitment and maturation develop into several mesenchymal phenotypes. Recently, we have described the presence of MSC in human cord blood (cbMSC) and informed that their properties are the same as those for MSC obtained from adult bone marrow. In this study we have investigated the capability of transplanted cbMSC to home and survive in the marrow of unconditioned nude mice. cbMSC utilized for transplantation studies were characterized by morphology, differentiation potential, and immunophenotype. After transplantation by systemic infusion, human DNA (as detected by PCR amplification of human-specific beta-globin gene) was detected in the marrow of recipients as well as in ex vivo-expanded stromal cells prepared from the marrow of transplanted animals. These results demonstrate homing and survival of cbMSC into the recipient marrow and also suggest a mesenchymal-orientated fate of engrafted cells, because human DNA was also detected in cells of other recipient tissues, like cardiac muscle, teeth, and spleen.

Mesenchymal stem cell therapy for the treatment of amyotrophic lateral sclerosis: signals for hope?

Based on the distinctive cellular, molecular and immunomodulatory traits of mesenchymal stem cells (MSC), it has been postulated that these cells may play a critical role in regenerative medicine. In addition to the participation of MSC in the repair of mesodermal-derived tissues (bone, cartilage), robust data have suggested that MSC may also play a reparative role in conditions involving damage of cells of ectodermal origin. The above content has been supported by the capability of MSC to differentiate into neuron-like cells as well as by a competence to generate a 'neuroprotective' environment. In turn, several preclinical studies have put forward the concept that MSC therapy may represent an option for the treatment of several neurological disorders and injuries, including amyotrophic lateral sclerosis. We expect that the above foundations, which have inspired this review, may result in the founding of an effective and/or palliative therapy for amyotrophic lateral sclerosis.

Mesenchymal stem cells and the treatment of conditions and diseases: the less glittering side of a conspicuous stem cell for basic research.

Not too long ago, several motivated and forward-looking articles were published describing the cellular and molecular properties of mesenchymal stem cells (MSCs), specially highlighting their potential for self-renewal, commitment, differentiation, and maturation into specific mesoderm-derived lineages. A very influential publication of that period entitled "Mesenchymal stem cells: No longer second class marrow citizens" [1] raised the point of view that "…challenges to harness MSC cell therapy to treat diseases … need to wait for the full comprehension that marrow is a rich source of mesenchyme-derived cells whose potential is still far from fully appreciated." Whether or not the prophecy of Gerson was fulfilled, in the last 8 years it has become evident that infusing MSCs into patients suffering a variety of disorders represents a viable option for medical treatment. Accordingly, a vast number of articles have explored the privileged cellular and molecular features of MSCs prepared from sources other than the canonical, represented by the bone marrow. This review will provide more information neither related to the biological attractiveness of MSCs nor to the success after their clinical use. Rather, we would like to underscore several "critical and tangential" issues, not always discussed in biomedical publications, but relevant to the clinical utilization of bone-marrow-derived MSCs.

Therapeutic angiogenesis in patients with severe limb ischemia by transplantation of a combination stem cell product.

OBJECTIVE: Angiogenesis involves the interplay of endothelial progenitor cells, pericytes, growth factors, and cellular matrix components. The use of mesenchymal stem cells, which are closely related to pericytes and produce diverse angiogenic growth factors and matrix molecules, seems to be a promising therapeutic modality. We postulate that the use of a combination cell product (mesenchymal stem cells in conjunction with a source of endothelial progenitor cells) is safe and efficient and may optimize the clinical results obtained with the use of endothelial progenitor cells alone. This study assessed whether the intramuscular infusion of a combination cell product represents a viable, effective, and lasting therapeutic modality to improve perfusion in severely ischemic limbs. METHODS: Patients with limb ischemia (n=26) received an intramuscular (gastrocnemius) infusion of the combination cell product in the most ischemic leg and a placebo product in the (less ischemic) contralateral leg. Clinical follow-up (months 0.5, 1, 2, and 4 postinfusion) included evaluation of pain-free walking time, ankle-brachial index, perfusion scintigraphy, and quality of life survey. RESULTS: No adverse events occurred after infusion. Efficacy assessment indicated that after cell infusion there was a significant improvement in walking time and ankle-brachial index. In addition, technetium-99m-tetrofosmin scintigraphy demonstrated a significant increase of perfusion in the treated limbs compared with the respective control legs. CONCLUSIONS: This phase II clinical trial shows that the use of a combination cell therapy is safe and effective in increasing blood flow in the ischemic legs of patients with limb ischemia.

Myocardial implantation of a combination stem cell product by using a transendocardial MYOSTAR injection catheter: A technical assessment.

AIM: Different types of progenitor cells have been used to improve cardiac conditions after myocardial infarction (MI). Results have shown that while the infusion of a single cell type is safe and feasible, efficacy is modest. Recently, the use of a combination, rather than a single, stem cell product has emerged as an attractive option to improve cardiac outcome after a MI. Before initiating a phase II clinical trial to assess safety and efficacy after the transendocardial infusion of a combination stem cell product, a bench testing assay was designed to validate that delivery through the injection catheter is not associated with cell loss/damage. The latter is important since mesenchymal stem cells (MSC), a component of the cell product, consist of large cells expressing matrix molecules and adhesive receptors. METHODS: The cell product (a mixture of mononuclear cells and MSC) was sequentially injected through a Myostar injection catheter. Exiting fractions were assessed for cell number, viability, capability to restart cell growth and immunophenotype. RESULTS: Cell recovery and viability were high. In turn, exiting cells preserved their biological properties and immunophenotype. CONCLUSIONS: Delivery of cells through a Myostar catheter is safe and not associated with changes in cell survival and/or properties.

Combination stem cell therapy for the treatment of medically refractory coronary ischemia: a Phase I study.

PURPOSE: Infusion of a source of endothelial progenitor cells (EPC) into the ischemic myocardium is emerging as a promising therapy for coronary ischemia, probably mediated by the formation of new blood vessels. Studies have shown that while the procedure is safe and feasible, efficacy results are contentious. The investigators hypothesized that the infusion of a combination cell product consisting of a source of EPC and mesenchymal stem cells (MSC) is safe and promotes the formation of more stable and mature blood vessels resulting in improved clinical outcomes. METHODS: Ten patients with stable angina pectoris (class III to IV) on maximal medical therapy were included. All patients had ≥ 70% stenosis in at least one coronary artery, and none was considered a candidate for percutaneous coronary intervention or coronary artery bypass graft. End points were feasibility and safety of intracoronary infusion of the combination cell product and assessment of myocardial ischemia, left ventricular ejection fraction (LVEF), and quality of life at 6 months postinfusion. RESULTS: Six months after cell infusion there were no adverse clinical events. Functional cardiac evaluation during the same period showed significant improvements in LVEF (average increase: 11%, P = .02) and myocardial ischemia (average decrease: 1.8 fold, P = .02). Additionally, all patients described significant improvements in quality of life. CONCLUSIONS: Despite the inherent limitations associated with a Phase I clinical trial, this study demonstrates that the intracoronary infusion of the combination cell product is feasible and safe and also insinuates that this form of therapy may be beneficial.

Combination stem cell therapy for the treatment of severe limb ischemia: safety and efficacy analysis.

The infusion of a source of endothelial progenitors (EPCs) to limb ischemia (LB) patients has been used to increase angiogenesis. Because the formation of new blood vessels involves, in addition to EPCs, other cells and angiogenic regulators, we postulate that a combination cell therapy including EPCs and mesenchymal stem cells (a source of pericytes progenitors and angiogenic regulators) may represent a preferential stimuli for the development of blood vessels. In this phase I clinical trial, patients with LI were infused with a cell product consisting of autologous bone marrow-derived mononuclear and mesenchymal stem cells. After 10 2 months of follow-up, efficacy assessment demonstrated improvements in walking time, ankle brachial pressure, and quality of life. Concomitantly, angiographic and 99mTc-TF perfusion scintigraphy scores confirmed increased perfusion in the treated limbs. These results show that the use of a combination cell therapy is safe, feasible, and appears effective in patients with LI.