Bone marrow mesenchymal stem cell niches and regenerative medicine

Mesenchymal progenitors are a powerful tool in regenerative medicine, but suffer from a rapid loss of differentiation potential during in vitro expansion. The recent discovery that well-characterized stem cells, like HSC, maintain their stemness during self-renewal through the interaction with specialized microenvironments, called stem cell niches, prompted us to investigate the existence of a niche compartment for also mesenchymal progenitors. In Chapter 4 of this thesis we described the establishment of a niche/progenitor system in vitro for bone marrow mesenchymal stem cells (MSC). We asked whether the non-adherent fraction of human bone marrow cultures contained early progenitors which can constitute a reservoir for the mesenchymal compartment and whether the adherent cells, instead, could provide a niche function for the maintenance and regulation of these progenitors. Replating the non-adherent fraction in a new dish at the first medium change, we found that a population of bone marrow non-adherent mesenchymal progenitors (BM-NAMP) was present and their number was 20.43.6% of the initial CFU-f. However, further investigation showed that, when serially replated in new dishes, BM-NAMP were able to steadily increase in number, self-renewing as non-adherent progenitors while generating at the same time adherent colonies. The diameter size evaluation showed that BM-NAMP could produce colonies with 2-fold larger diameter, indicating a significantly higher proliferation capacity. However, the colonies produced in the following replating steps were progressively smaller, indicating a gradual loss of BM-NAMP proliferation potential. Together with increased proliferation, first-replated BM-NAMP progeny cells displayed a higher differentiation potential compared to standard CFU-f both in vitro and in vivo. Taken together, these data indicate together that BM-NAMP show features of earlier progenitor features and suggest a biological difference between BM-NAMP and the initially adhrering CFU-f. Serial replating experiments performed with serum alone showed that BM-NAMP critically required FGF-2 for their initial selection and maintenance in culture. Interestingly, blocking receptor experiments showed that the maintenance of BM-NAMP in culture was mediated through FGFR2c signaling, which has been shown to be involved in vivo in the balance between proliferation and differentiation of skeletal progenitors. We also hypothesized that BM-NAMP were in close interaction with the adherent cells, and that these provide a niche function for them. BM-NAMP were not able to survive when replated either on agarose-coated dishes or on human fibroblasts. This suggests that BM-NAMP required specific signals from the adherent progeny and that this fraction constitutes a unique environment for BM-NAMP survival and self-renewal. In fact, when kept in contact with initial CFU-f progeny for 14 days instead of being serially replated, BM-NAMP were able to produce 3-fold more colonies. Furthermore, the colony diameter analysis showed that, unlike the serial replating which caused a gradual loss of BM-NAMP proliferative activity, the continuous culture in the primary plate could preserve BM-NAMP proliferation potential. Furthermore, if kept in the original plate, BM-NAMP could generate a progeny that also displayed a higher differentiation capacity. Taken together, these results suggest together that CFU-f progeny provides a niche function for BM-NAMP. In Chapter 5 we sought at investigating the presence of a class of non-adherent progenitors in human adipose tissue stromal vascular fraction (SVF), which constitute an abundant source of mesenchymal progenitors, to determine whether the NAMP compartment was specific to bone marrow or they could constitute a reservoir also in other tissues. NAMP were present in adipose tissue SVF cultures (AT-NAMP) with a similar frequency as observed in the bone marrow and the replating of the non-adherent fraction in the same dish revealed that they were stably non-adherent. The main difference compared to BM-NAMP was the inability of AT-NAMP to self-renew as non-adherent progenitors upon serial replating, since only few colonies were present in the last replating step. However, these colonies had a significantly increased diameter. This suggests that, when serially replated, AT-NAMP do not undergo proliferation but rather a selection for the very rare progenitors with the highest proliferation ability. Similarly to BM-NAMP, when kept in contact with the initially adhering CFU-f, AT-NAMP could proliferate without loss of their proliferation capacity. This suggests that, as for bone marrow cells, adherent CFU-f provide a niche function for the non-adherent progenitors, regulating the maintenance of their early-progenitor properties. In conclusion, these data show that, although displaying important tissue-specific biological differences, NAMP are present in the mesenchymal progenitor compartment of different tissues and they represent a reservoir of earlier progenitors compared to standard CFU-f

It Takes Two to Tang: Coupling of Angiogenesis and Osteogenesis for Bone Regeneration

Bone regeneration is a complex process requiring highly orchestrated interactions between different cells and signals to form new mineralized tissue. Blood vessels serve as a structural template, around which bone development takes place, and also bring together the key elements for bone homeostasis into the osteogenic microenvironment, including minerals, growth factors and osteogenic progenitor cells. Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth and it is required for effective coupling of angiogenesis and osteogenesis during both skeletal development and postnatal bone repair. Here, we will review the current state of knowledge on the molecular cross-talk between angiogenesis and osteogenesis. In particular, we will focus on the role of VEGF in coupling these two processes and how VEGF dose can control the outcome, addressing in particular: (1) the direct influence of VEGF on osteogenic differentiation of mesenchymal progenitors; (2) the angiocrine functions of endothelium to regulate osteoprogenitors; (3) the role of immune cells, e.g., myeloid cells and osteoclast precursors, recruited by VEGF to the osteogenic microenvironment. Finally, we will discuss emerging strategies, based on the current biological understanding, to ensure rapid vascularization and efficient bone formation in regenerative medicine

Semaphorin 3A promotes the long-term persistence of human SVF-derived microvascular networks in engineered grafts

Introduction: The stromal vascular fraction (SVF) of human adipose tissue is an attractive cell source for engineering grafts with intrinsic vascularization potential, as it is rich in vasculogenic progenitors. However, in order to maintain their functional perfusion it is important to promote the in vivo stabilization of newly assembled microvascular networks. We previously found that Semaphorin 3A (Sema3A) promotes the rapid stabilization of new blood vessels induced by VEGF overexpression in skeletal muscle. Here we investigated whether Sema3A could promote the assembly, connection to circulation and persistence of human SVF-derived microvascular networks in engineered grafts. Methods: Recombinant Sema3A was engineered with a transglutaminase substrate sequence (TG-Sema3A) to allow cross-linking into fibrin hydrogels. Grafts were prepared with freshly isolated human SVF cells in fibrin hydrogels decorated with 0, 0.1 or 100 μg/ml TG-Sema3A and implanted subcutaneously in immune-deficient mice. Results: After 1 week in vivo, the assembly of human-derived networks was similar in all conditions. The outer part of the grafts was populated by blood vessels of both human and mouse origin, which formed abundant hybrid structures within a common basal lamina. About 90% of human-derived blood vessels were functionally connected to the host circulation in all conditions. However, in the control samples human vessels were unstable. In fact, they significantly regressed by 6 weeks and could no longer be found by 12 weeks. In contrast, a low Sema3A dose (0.1 μg/ml) promoted further human vascular expansion by about 2-fold at 6 weeks and protected them from regression until 12 weeks. From a mechanistic point of view, the stabilization of SVF-derived vessels by 0.1 μg/ml of Sema3A correlated with the recruitment of a specific population of monocytes expressing its receptor Neuropilin-1. Discussion: In conclusion, Sema3A is a potent stimulator of in vivo long-term persistence of microvascular networks derived from human SVF. Therefore, decoration of matrices with Sema3a can be envisioned to promote the functional support of tissue engineered grafts.</p

VEGF Over-Expression by Engineered BMSC Accelerates Functional Perfusion, Improving Tissue Density and In-Growth in Clinical-Size Osteogenic Grafts

The first choice for reconstruction of clinical-size bone defects consists of autologous bone flaps, which often lack the required mechanical strength and cause significant donor-site morbidity. We have previously developed biological substitutes in a rabbit model by combining bone tissue engineering and flap pre-fabrication. However, spontaneous vascularization was insufficient to ensure progenitor survival in the core of the constructs. Here, we hypothesized that increased angiogenic stimulation within constructs by exogenous VEGF can significantly accelerate early vascularization and tissue in-growth. Bone marrow stromal cells from NZW rabbits (rBMSC) were transduced with a retroviral vector to express rabbit VEGF linked to a truncated version of rabbit CD4 as a cell-surface marker. Autologous cells were seeded in clinical-size 5.5 cm; 3; HA scaffolds wrapped in a panniculus carnosus flap to provide an ample vascular supply, and implanted ectopically. Constructs seeded with VEGF-expressing rBMSC showed significantly increased progenitor survivival, depth of tissue ingrowth and amount of mineralized tissue. Contrast-enhanced MRI after 1 week; in vivo; showed significantly improved tissue perfusion in the inner layer of the grafts compared to controls. Interestingly, grafts containing VEGF-expressing rBMSC displayed a hierarchically organized functional vascular tree, composed of dense capillary networks in the inner layers connected to large-caliber feeding vessels entering the constructs at the periphery. These data constitute proof of principle that providing sustained VEGF signaling, independently of cells experiencing hypoxia, is effective to drive rapid vascularization and increase early perfusion in clinical-size osteogenic grafts, leading to improved tissue formation deeper in the constructs

VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-β1 paracrine axis

VEGF is widely investigated for therapeutic angiogenesis, but while short-term delivery is desirable for safety, it is insufficient for new vessel persistence, jeopardizing efficacy. Here, we investigated whether and how VEGF dose regulates nascent vessel stabilization, to identify novel therapeutic targets. Monoclonal populations of transduced myoblasts were used to homogeneously express specific VEGF doses in SCID mouse muscles. VEGF was abrogated after 10 and 17 days by Aflibercept treatment. Vascular stabilization was fastest with low VEGF, but delayed or prevented by higher doses, without affecting pericyte coverage. Rather, VEGF dose-dependently inhibited endothelial Semaphorin3A expression, thereby impairing recruitment of Neuropilin-1-expressing monocytes (NEM), TGF-β1 production and endothelial SMAD2/3 activation. TGF-β1 further initiated a feedback loop stimulating endothelial Semaphorin3A expression, thereby amplifying the stabilizing signals. Blocking experiments showed that NEM recruitment required endogenous Semaphorin3A and that TGF-β1 was necessary to start the Semaphorin3A/NEM axis. Conversely, Semaphorin3A treatment promoted NEM recruitment and vessel stabilization despite high VEGF doses or transient adenoviral delivery. Therefore, VEGF inhibits the endothelial Semaphorin3A/NEM/TGF-β1 paracrine axis and Semaphorin3A treatment accelerates stabilization of VEGF-induced angiogenesis

Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation

Risk for cancer development in familial Mediterranean fever and associated predisposing factors: an ambidirectional cohort study from the international AIDA Network registries

Objective: Inflammation has been associated with an increased risk for cancer development, while innate immune system activation could counteract the risk for malignancies. Familial Mediterranean fever (FMF) is a severe systemic inflammatory condition and also represents the archetype of innate immunity deregulation. Therefore, the aim of this study is to investigate the risk for cancer development in FMF. Methods: The risk ratio (RR) for malignancies was separately compared between FMF patients and fibromyalgia subjects, Still's disease patients and Behçet's disease patients. Clinical variables associated with cancer development in FMF patients were searched through binary logistic regression. Results: 580 FMF patients and 102 fibromyalgia subjects, 1012 Behçet's disease patients and 497 Still's disease patients were enrolled. The RR for the occurrence of malignant neoplasms was 0.26 (95% Confidence Interval [CI.] 0.10-0.73, p=0.006) in patients with FMF compared to fibromyalgia subjects; the RR for the occurrence of malignant cancer was 0.51 (95% CI. 0.23-1.16, p=0.10) in FMF compared to Still's disease and 0.60 (95% CI. 0.29-1.28, p=0.18) in FMF compared to Behçet's disease. At logistic regression, the risk of occurrence of malignant neoplasms in FMF patients was associated with the age at disease onset (β1 = 0.039, 95% CI. 0.001-0.071, p=0.02), the age at the diagnosis (β1 = 0.048, 95% CI. 0.039-0.085, p=0.006), the age at the enrolment (β1 = 0.05, 95% CI. 0.007-0.068, p=0.01), the number of attacks per year (β1 = 0.011, 95% CI. 0.001- 0.019, p=0.008), the use of biotechnological agents (β1 = 1.77, 95% CI. 0.43-3.19, p=0.009), the use of anti-IL-1 agents (β1 = 2.089, 95% CI. 0.7-3.5, p=0.002). Conclusions: The risk for cancer is reduced in Caucasic FMF patients; however, when malignant neoplasms occur, this is more frequent in FMF cases suffering from a severe disease phenotype and presenting a colchicine-resistant disease

Risk for cancer development in familial Mediterranean fever and associated predisposing factors: an ambidirectional cohort study from the international AIDA Network registries

Objective: Inflammation has been associated with an increased risk for cancer development, while innate immune system activation could counteract the risk for malignancies. Familial Mediterranean fever (FMF) is a severe systemic inflammatory condition and also represents the archetype of innate immunity deregulation. Therefore, the aim of this study is to investigate the risk for cancer development in FMF. Methods: The risk ratio (RR) for malignancies was separately compared between FMF patients and fibromyalgia subjects, Still's disease patients and Beh &amp; ccedil;et's disease patients. Clinical variables associated with cancer development in FMF patients were searched through binary logistic regression. Results: 580 FMF patients and 102 fibromyalgia subjects, 1012 Beh &amp; ccedil;et's disease patients and 497 Still's disease patients were enrolled. The RR for the occurrence of malignant neoplasms was 0.26 (95% Confidence Interval [CI.] 0.10-0.73, p=0.006) in patients with FMF compared to fibromyalgia subjects; the RR for the occurrence of malignant cancer was 0.51 (95% CI. 0.23-1.16, p=0.10) in FMF compared to Still's disease and 0.60 (95% CI. 0.29-1.28, p=0.18) in FMF compared to Beh &amp; ccedil;et's disease. At logistic regression, the risk of occurrence of malignant neoplasms in FMF patients was associated with the age at disease onset (beta 1 = 0.039, 95% CI. 0.001-0.071, p=0.02), the age at the diagnosis (beta 1 = 0.048, 95% CI. 0.039-0.085, p=0.006), the age at the enrolment (beta 1 = 0.05, 95% CI. 0.007-0.068, p=0.01), the number of attacks per year (beta 1 = 0.011, 95% CI. 0.001- 0.019, p=0.008), the use of biotechnological agents (beta 1 = 1.77, 95% CI. 0.43-3.19, p=0.009), the use of anti-IL-1 agents (beta 1 = 2.089, 95% CI. 0.7-3.5, p=0.002).Conclusions: The risk for cancer is reduced in Caucasic FMF patients; however, when malignant neoplasms occur, this is more frequent in FMF cases suffering from a severe disease phenotype and presenting a colchicine-resistant disease

Clinical and laboratory features associated with macrophage activation syndrome in Still's disease: data from the international AIDA Network Still's Disease Registry

: To characterize clinical and laboratory signs of patients with still's disease experiencing macrophage activation syndrome (MAS) and identify factors associated with MAS development. patients with still's disease classified according to internationally accepted criteria were enrolled in the autoInflammatory disease alliance (AIDA) still's disease registry. clinical and laboratory features observed during the inflammatory attack complicated by MAS were included in univariate and multivariate logistic regression analysis to identify factors associated to MAS development. A total of 414 patients with Still's disease were included; 39 (9.4%) of them developed MAS during clinical history. At univariate analyses, the following variables were significantly associated with MAS: classification of arthritis based on the number of joints involved (p = 0.003), liver involvement (p = 0.04), hepatomegaly (p = 0.02), hepatic failure (p = 0.01), axillary lymphadenopathy (p = 0.04), pneumonia (p = 0.03), acute respiratory distress syndrome (p &lt; 0.001), platelet abnormalities (p &lt; 0.001), high serum ferritin levels (p = 0.009), abnormal liver function tests (p = 0.009), hypoalbuminemia (p = 0.002), increased LDH (p = 0.001), and LDH serum levels (p &lt; 0.001). at multivariate analysis, hepatomegaly (OR 8.7, 95% CI 1.9-52.6, p = 0.007) and monoarthritis (OR 15.8, 95% CI 2.9-97.1, p = 0.001), were directly associated with MAS, while the decade of life at Still's disease onset (OR 0.6, 95% CI 0.4-0.9, p = 0.045), a normal platelet count (OR 0.1, 95% CI 0.01-0.8, p = 0.034) or thrombocytosis (OR 0.01, 95% CI 0.0-0.2, p = 0.008) resulted to be protective. clinical and laboratory factors associated with MAS development have been identified in a large cohort of patients based on real-life data

A familiar stranger: CD34 expression and putative functions in SVF cells of adipose tissue

Human adipose tissue obtained by liposuction is easily accessible and an abundant potential source of autologous cells for regenerative medicine applications. After digestion of the tissue and removal of differentiated adipocytes, the so-called stromal vascular fraction (SVF) of adipose, a mix of various cell types, is obtained. SVF contains mesenchymal fibroblastic cells, able to adhere to culture plastic and to generate large colonies in vitro, that closely resemble bone marrow-derived colony forming units-fibroblastic, and whose expanded progeny, adipose mesenchymal stem/stromal cells (ASC), show strong similarities with bone marrow mesenchymal stem cells. The sialomucin CD34, which is well known as a hematopoietic stem cell marker, is also expressed by ASC in native adipose tissue but its expression is gradually lost upon standard ASC expansion in vitro. Surprisingly little is known about the functional role of CD34 in the biology and tissue forming capacity of SVF cells and ASC. The present editorial provides a short introduction to the CD34 family of sialomucins and reviews the data from the literature concerning expression and function of these proteins in SVF cells and their in vitro expanded progeny