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Introduction: Acute myeloid leukemia (AML) is a highly heterogeneous malignancy caused by various genetic alterations and characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). This abnormal growth of AML cells disrupts normal hematopoiesis and alters the BM microenvironment components, establishing a niche supportive of leukemogenesis. Bone marrow stromal cells (BMSCs) play a pivotal role in giving rise to essential elements of the BM niche, including adipocytes and osteogenic cells. Animal models have shown that the BM microenvironment is significantly remodeled by AML cells, which skew BMSCs toward an ineffective osteogenic differentiation with an accumulation of osteoprogenitors. However, little is known about the mechanisms by which AML cells affect osteogenesis., Methods: We studied the effect of AML cells on the osteogenic commitment of normal BMSCs, using a 2D co-culture system., Results: We found that AML cell lines and primary blasts, but not normal hematopoietic CD34 + cells, induced in BMSCs an ineffective osteogenic commitment, with an increase of the early-osteogenic marker tissue non-specific alkaline phosphatase (TNAP) in the absence of the late-osteogenic gene up-regulation. Moreover, the direct interaction of AML cells and BMSCs was indispensable in influencing osteogenic differentiation. Mechanistic studies identified a role for AML-mediated Notch activation in BMSCs contributing to their ineffective osteogenic commitment. Inhibition of Notch using a γ-secretase inhibitor strongly influenced Notch signaling in BMSCs and abrogated the AML-induced TNAP up-regulation., Discussion: Together, our data support the hypothesis that AML infiltration produces a leukemia-supportive pre-osteoblast-rich niche in the BM, which can be partially ascribed to AML-induced activation of Notch signaling in BMSCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tomasoni, Arsuffi, Donsante, Corsi, Riminucci, Biondi, Pievani and Serafini.)

Mesenchymal stromal cells (MSCs) represent an essential component of the bone marrow (BM) niche and display disease-specific alterations in several myeloid malignancies. The aim of this work was to study possible MSC abnormalities in Philadelphia-negative myeloproliferative neoplasms (MPNs) in relationship to the degree of BM fibrosis. MSCs were isolated from BM of 6 healthy donors (HD) and of 23 MPN patients, classified in 3 groups according to the diagnosis and the grade of BM fibrosis: polycythemia vera and essential thrombocythemia (PV/ET), low fibrosis myelofibrosis (LF-MF), and high fibrosis MF (HF-MF). MSC cultures were established from 21 of 23 MPN patients. MPN-derived MSCs did not exhibit any functional impairment in their adipogenic/osteogenic/chondrogenic differentiation potential and displayed a phenotype similar to HD-derived MSCs but with a decreased expression of CD146. All MPN-MSC lines were negative for the patient-specific hematopoietic clone mutations (JAK2, MPL, CALR). MSCs derived from HF-MF patients displayed a reduced clonogenic potential and a lower growth kinetic compared to MSCs from HD, LF-MF, and PV/ET patients. mRNA levels of hematopoiesis regulatory molecules were unaffected in MSCs from HF-MF compared to HD. Finally, in vitro ActivinA secretion by MSCs was increased in HF-MF compared to LF-MF patients, in association with a lower hemoglobin value. Increased ActivinA immunolabeling on stromal cells and erythroid precursors was also observed in HF-MF BM biopsies. In conclusion, higher grade of BM fibrosis is associated with functional impairment of MSCs and the increased secretion of ActivinA may represent a suitable target for anemia treatment in MF patients.

Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") contain a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). It is now apparent that cells with similar but not identical properties can be isolated from other skeletal compartments (growth plate, periosteum). The biological properties of BMSCs, and these related stem/progenitor cells, are assessed by a variety of assays, both in vitro and in vivo. Application of these assays in an appropriate fashion provide a great deal of information on the role of BMSCs, and the subset of SSCs, in health and in disease.

Effects of oxygen tension on the generation, expansion, proliferation and differentiation of stromal cell types is widely described in the literature. However, data on the internal heterogeneity of applied cell populations at different O 2 levels and possible impacts on differentiation potentials are controversial. Here, the expression of 39 human HOX genes was determined in neonatal cord blood stromal cells and linked to differentiation-associated signatures. In cord blood, unrestricted somatic stromal cells (USSCs), lacking HOX gene expression, and cord blood-derived multipotent stromal cells (CB-MSCs), expressing about 20 HOX genes, are distinguished by their specific HOX code. Interestingly, 74% of the clones generated at 21% O 2 were HOX-negative USSCs, whereas 73% of upcoming clones at 3% O 2 were HOX-positive CB-MSCs. In order to better categorize distinct cell lines generated at 3% O 2 , the expression of all 39 HOX genes within HOX clusters A, B, C and D were tested and new subtypes defined: cells negative in all four HOX clusters (USSCs); cells positive in all four clusters (CB-MSCs ABCD ); and subpopulations missing a single cluster (CB-MSCs ACD and CB-MSCs BCD ). Comprehensive qPCR analyses of established chondro-osteomarkers revealed subtype-specific signatures verifiably associated with in vitro and in vivo differentiation capacity. The data presented here underline the necessity of better characterizing distinct cell populations at a clonal level, taking advantage of the inherent specific HOX code as a distinguishing feature between individual subtypes. Moreover, the correlation of subtype-specific molecular signatures with in vitro and in vivo bone formation is discussed. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

A widely shared view reads that mesenchymal stem/stromal cells ("MSCs") are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as "MSCs" based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called "MSCs," with important applicative implications. The data also support the view that rather than a uniform class of "MSCs," different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background Aims: Cord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated., Methods: MSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan., Results: We were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan., Conclusions: Our results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") can establish the hematopoietic microenvironment within heterotopic ossicles generated by transplantation at non-skeletal sites. Here we show that non-mineralized cartilage pellets formed by hBMSCs ex vivo generate complete ossicles upon heterotopic transplantation in the absence of exogenous scaffolds. These ossicles display a remarkable degree of architectural fidelity, showing that an exogenous conductive scaffold is not an absolute requirement for bone formation by transplanted BMSCs. Marrow cavities within the ossicles include erythroid, myeloid and granulopoietic lineages, clonogenic hematopoietic progenitors and phenotypic HSCs, indicating that complete stem cell niches and hematopoiesis are established. hBMSCs (CD146(+) adventitial reticular cells) are established in the heterotopic chimeric bone marrow through a unique process of endochondral bone marrow formation, distinct from physiological endochondral bone formation. In this process, chondrocytes remain viable and proliferate within the pellet, are released from cartilage, and convert into bone marrow stromal cells. Once explanted in secondary culture, these cells retain phenotype and properties of skeletal stem cells ("MSCs"), including the ability to form secondary cartilage pellets and secondary ossicles upon serial transplantation. Ex vivo, hBMSCs initially induced to form cartilage pellets can be reestablished in adherent culture and can modulate gene expression between cartilage and stromal cell phenotypes. These data show that so-called "cartilage differentiation" of BMSCs in vitro is a reversible phenomenon, which is actually reverted, in vivo, to the effect of generating stromal cells supporting the homing of hematopoietic stem cells and progenitors., (Copyright © 2014. Published by Elsevier B.V.)

Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") contain a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). The biological properties of BMSC cultures are assessed by a variety of assays, both in vitro and in vivo. Application of these assays in an appropriate fashion provides a great deal of information on the role of BMSCs, and the subset of SSCs, in health and in disease.

The identification of skeletal progenitor cells in the human bone marrow (so-called mesenchymal stem cells) by anatomy and phenotype (CD146-expressing, adventitial reticular cells) has coincided with the recognition that the ability to transfer the hematopoietic microenvironment is an inherent property of skeletal progenitor cells. Inasmuch as these cells generate osteoblasts, associate with sinusoids (the assembly of which they dynamically direct), and coincide with, and self-renew into, stromal reticular cells, these cells are pivotal organizers of the hematopoietic microenvironment. Their nature as osteogenic cells and sinusoidal location reconcile the dual view of endosteal surfaces and sinusoidal walls as the hematopoietic stem cell "niches", and highlight the dynamic nature of a niche/microenvironment essentially maintained by cells with properties of progenitors/stem cells for skeletal tissues. This view brings the long recognized, and somewhat mysterious, interaction between bone and bone marrow into a new perspective, where two stem cells interact with each other at the same niche., (Copyright © 2011. Published by Elsevier Ltd.)

At the turn of a decade of intensive wishful thinking, "mesenchymal stem cells" are changing their profile, while retaining their charm. As hopes to turn bone into brain or vice versa seem on the wane, we learn (1) that the archetypal "mesenchymal stem cell," the skeletal stem cell found in the bone marrow, can be directly identified as a specialized type of mural cell/pericyte, found in the wall of sinusoids and long known as adventitial reticular cells; (2) that bone marrow skeletal stem cells are also defined by expression of CD146, and can self-renew in vivo, while giving rise to skeletal tissues, and therefore earn consideration as bona fide stem cells; (3) that a broader class of microvascular mural cells endowed with clonogenicity and progenitor properties may exist in other tissues, although their true potency needs to be firmly established by stringent assays and thorough comparisons across tissues; (4) that bone marrow skeletal stem cells display unique angiopoietic and hematopoietic niche-related functions, consisting in their ability to transfer the hematopoietic microenvironment and to guide the assembly of microvascular networks, which seem to define their inherent biology; and (5) that use of skeletal stem cells as disease models, and as models of high-risk strategies for cell and gene therapy specifically in incurable skeletal diseases, may provide new challenges for the next decade, and perhaps reward for medicine in the one that follows.

Objectives: Mesenchymal stromal cells (MSCs) have attracted considerable interest in both the scientific and clinical fields. In order to obtain a sufficient cell number for application, their in vitro expansion is necessary, but during this process their characteristics may be altered and cells may acquire oncogenic properties. We have investigated properties of MSC that may be related to oncogenesis, a critical parameter that has to be evaluated prior to MSC clinical use., Materials and Methods: We studied the expression of p53, p16, RB, H-RAS and human telomerase reverse transcriptase (hTERT) in MSCs from bone marrow of children diagnosed with idiopathic thrombocytopenic purpura (ITP) and autoimmune neutropenia. The same cells were seeded in soft agar to confirm their anchorage dependence and were karyotypically analysed. Finally, MSCs were subcutaneously transplanted into SCID mice and their ectopic osteogenic as well as tumorigenic potential was evaluated., Results: We have shown that MSCs derived from bone marrow of children with ITP and autoimmune neutropenia do not undergo transformation, the cells expressed normal levels of p53, p16, RB and H-RAS. Expression of hTERT was undetectable, chromosome content remained stable, and their anchorage dependence was confirmed. In an in vivo model, when MSCs were subcutaneously transplanted into SCID mice, no tumorigenesis was observed., Conclusions: These findings suggest that MSCs from bone marrow of children do not have oncogenic properties and, therefore, represent validate candidates for applications in regenerative medicine.

Expression of "stemness" markers is widely used as a predictor of stem cell properties of mesenchymal stem cells (MSC). Here, we show that bone marrow-derived (BM)-MSC show stem cell-like behavior in vivo; that is, they form ossicles with formation of bone, formation of adipocytes, and establishment of the murine hematopoietic microenvironment. Multipotent umbilical vein-derived stromal cells (UVSC), on the other hand, do not form bone, nor do they give rise to adipocytes in vivo. Despite these differences in stem-cell-like behavior, BM-MSC and UVSC express the two transcripts variants of POU5F1 at a similar level. Also, we found that in BM-MSC and UVSC, POU5F1 is detectable. However, more than 89% of the POU5F1 transcripts correspond to the POU5F1P1, -P3, or -P4 pseudogene. Despite low-level expression of POU5F1, we were unable to precipitate POU5F1 protein in either BM-MSC or UVSC. These results demonstrate that MSC stemness does not correlate to expression of POU5F1 transcripts or its pseudogenes.

The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC "niche" involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.

Stem cells have become a major area of interest in the treatment of human disease, but more recently, stem cells have come to be appreciated as the cause of disease. Fibrous dysplasia of bone and the McCune-Albright Syndrome evolve from activating missense mutations in Gsalpha in pluripotent embryonic stem cells. The legacy of these mutations remains in a population of mutated multipotent post-natal skeletal stem cells ("mesenchymal" stem cells), which direct the formation of abnormal bone and a fibrotic marrow in fibrous dysplasia. Future therapeutic approaches for the treatment of fibrous dysplasia, the most significant component of the McCune-Albright Syndrome, will depend on a greater understanding of post-natal skeletal stem cell biology and how skeletal stem cells can be manipulated for efficient bone regeneration.

Skeletal stem cells (SSCs, commonly referred to as "mesenchymal" stem cells) are found in the bone marrow stromal cell (BMSC) fraction of post-natal bone marrow. They can be isolated in culture as adherent, clonogenic cells endowed with the ability to grow and differentiate into multiple lineages, all of which correspond to tissues that are integral parts of the skeleton. The multipotency of SSCs is probed by in vivo transplantation assays. The ability of SSCs to generate a cell strain competent to form significant amounts of bone in vivo has led to the formulation of preclinical models of bone repair.

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.