Bone-forming perivascular cells: Cellular heterogeneity and use for tissue repair.

Mesenchymal progenitor cells are broadly distributed across perivascular niches-an observation conserved between species. One common histologic zone with a high frequency of mesenchymal progenitor cells within mammalian tissues is the tunica adventitia, the outer layer of blood vessel walls populated by cells with a fibroblastic morphology. The diversity and functions of (re)generative cells present in this outermost perivascular niche are under intense investigation; we have reviewed herein our current knowledge of adventitial cell potential with a somewhat narrow focus on bone formation. Antigens of interest to functionally segregate adventicytes are discussed, including CD10, CD107a, aldehyde dehydrogenase isoforms, and CD140a, among others. Purified adventicytes (such as CD10+ , CD107alow , and CD140a+ cells) have stronger osteogenic potential and promote bone formation in vivo. Recent bone tissue engineering applications of adventitial cells are also presented. A better understanding of perivascular progenitor cell subsets may represent a beneficial advance for future efforts in tissue repair and bioengineering.

A Role for MOSPD1 in Mesenchymal Stem Cell Proliferation and Differentiation.

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1-null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self-renewal capacity of MOSPD1-null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)-like cells expressing CD73, CD90, and CD105 were generated from MOSPD1-null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1-null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA-sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre-MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation.

Natural history of mesenchymal stem cells, from vessel walls to culture vessels.

Mesenchymal stem/stromal cells (MSCs) can regenerate tissues by direct differentiation or indirectly by stimulating angiogenesis, limiting inflammation, and recruiting tissue-specific progenitor cells. MSCs emerge and multiply in long-term cultures of total cells from the bone marrow or multiple other organs. Such a derivation in vitro is simple and convenient, hence popular, but has long precluded understanding of the native identity, tissue distribution, frequency, and natural role of MSCs, which have been defined and validated exclusively in terms of surface marker expression and developmental potential in culture into bone, cartilage, and fat. Such simple, widely accepted criteria uniformly typify MSCs, even though some differences in potential exist, depending on tissue sources. Combined immunohistochemistry, flow cytometry, and cell culture have allowed tracking the artifactual cultured mesenchymal stem/stromal cells back to perivascular anatomical regions. Presently, both pericytes enveloping microvessels and adventitial cells surrounding larger arteries and veins have been described as possible MSC forerunners. While such a vascular association would explain why MSCs have been isolated from virtually all tissues tested, the origin of the MSCs grown from umbilical cord blood remains unknown. In fact, most aspects of the biology of perivascular MSCs are still obscure, from the emergence of these cells in the embryo to the molecular control of their activity in adult tissues. Such dark areas have not compromised intents to use these cells in clinical settings though, in which purified perivascular cells already exhibit decisive advantages over conventional MSCs, including purity, thorough characterization and, principally, total independence from in vitro culture. A growing body of experimental data is currently paving the way to the medical usage of autologous sorted perivascular cells for indications in which MSCs have been previously contemplated or actually used, such as bone regeneration and cardiovascular tissue repair.

Lysosomal protein surface expression discriminates fat- from bone-forming human mesenchymal precursor cells.

Tissue resident mesenchymal stem/stromal cells (MSCs) occupy perivascular spaces. Profiling human adipose perivascular mesenchyme with antibody arrays identified 16 novel surface antigens, including endolysosomal protein CD107a. Surface CD107a expression segregates MSCs into functionally distinct subsets. In culture, CD107alow cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107ahigh cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107alow cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107ahigh cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107alow cells are precursors of CD107ahigh cells. These results document the molecular and functional diversity of perivascular regenerative cells, and show that relocation to cell surface of a lysosomal protein marks the transition from osteo- to adipogenic potential in native human MSCs, a population of substantial therapeutic interest.

Comparison of Human Tissue Microarray to Human Pericyte Transcriptome Yields Novel Perivascular Cell Markers.

Human perivascular progenitor cells, including pericytes, are well-described multipotent mesenchymal cells giving rise to mesenchymal stem cells in culture. Despite the unique location of pericytes, specific antigens to distinguish human pericytes from other cell types are few. Here, we employed a human tissue microarray (Human Protein Atlas) to identify proteins that are strongly and specifically expressed in a pericytic location within human adipose tissue. Next, these results were cross-referenced with RNA sequencing data from human adipose tissue pericytes, as defined as a fluorescence activated cell sorting (FACS) purified CD146+CD34-CD31-CD45- cell population. Results showed that from 105,532 core biopsies of soft tissue, 229 proteins showed strong and specific perivascular immunoreactivity, the majority of which (155) were present in the tunica intima. Next, cross-referencing with the transcriptome of FACS-derived CD146+ pericytes yielded 25 consistently expressed genes/proteins, including 18 novel antigens. A majority of these transcripts showed maintained expression after culture propagation (56% of genes). Interestingly, many novel antigens within pericytes are regulators of osteogenic differentiation. In sum, our study demonstrates the existence of novel pericyte markers, some of which are conserved in culture that may be useful for future efforts to typify, isolate, and characterize human pericytes.

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering.

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering.

Perivascular Stem Cells Diminish Muscle Atrophy Following Massive Rotator Cuff Tears in a Small Animal Model.

BACKGROUND: Rotator cuff tears are a common cause of shoulder pain and often necessitate operative repair. Muscle atrophy, fibrosis, and fatty infiltration can develop after rotator cuff tears, which may compromise surgical outcomes. This study investigated the regenerative potential of 2 human adipose-derived progenitor cell lineages in a murine model of massive rotator cuff tears. METHODS: Ninety immunodeficient mice were used (15 groups of 6 mice). Mice were assigned to 1 of 3 surgical procedures: sham, supraspinatus and infraspinatus tendon transection (TT), or TT and denervation via suprascapular nerve transection (TT + DN). Perivascular stem cells (PSCs) were harvested from human lipoaspirate and sorted using fluorescence-activated cell sorting into pericytes (CD146 CD34 CD45 CD31) and adventitial cells (CD146 CD34 CD45 CD31). Mice received no injection, injection with saline solution, or injection with pericytes or adventitial cells either at the time of the index procedure ("prophylactic") or at 2 weeks following the index surgery ("therapeutic"). Muscles were harvested 6 weeks following the index procedure. Wet muscle weight, muscle fiber cross-sectional area, fibrosis, and fatty infiltration were analyzed. RESULTS: PSC treatment after TT (prophylactic or therapeutic injections) and after TT + DN (therapeutic injections) resulted in less muscle weight loss and greater muscle fiber cross-sectional area than was demonstrated for controls (p < 0.05). The TT + DN groups treated with pericytes at either time point or with adventitial cells at 2 weeks postoperatively had less fibrosis than the TT + DN controls. There was less fatty infiltration in the TT groups treated with pericytes at either time point or with adventitial cells at the time of surgery compared with controls. CONCLUSIONS: Our findings demonstrated significantly less muscle atrophy in the groups treated with PSCs compared with controls. This suggests that the use of PSCs may have a role in the prevention of muscle atrophy without leading to increased fibrosis or fatty infiltration. CLINICAL RELEVANCE: Improved muscle quality in the setting of rotator cuff tears may increase the success rates of surgical repair and lead to superior clinical outcomes.

Stem cell technology for bone regeneration: current status and potential applications.

Continued improvements in the understanding and application of mesenchymal stem cells (MSC) have revolutionized tissue engineering. This is particularly true within the field of skeletal regenerative medicine. However, much remains unknown regarding the native origins of MSC, the relative advantages of different MSC populations for bone regeneration, and even the biologic safety of such unpurified, grossly characterized cells. This review will first summarize the initial discovery of MSC, as well as the current and future applications of MSC in bone tissue engineering. Next, the relative advantages and disadvantages of MSC isolated from distinct tissue origins are debated, including the MSC from adipose, bone marrow, and dental pulp, among others. The perivascular origin of MSC is next discussed. Finally, we briefly comment on pluripotent stem cell populations and their possible application in bone tissue engineering. While continually expanding, the field of MSC-based bone tissue engineering and regeneration shows potential to become a clinical reality in the not-so-distant future.

STR melting curve analysis as a genetic screening tool for crime scene samples.

In this proof-of-concept study, high-resolution melt curve (HRMC) analysis was investigated as a postquantification screening tool to discriminate human CSF1PO and THO1 genotypes amplified with mini-STR primers in the presence of SYBR Green or LCGreen Plus dyes. A total of 12 CSF1PO and 11 HUMTHO1 genotypes were analyzed on the LightScanner HR96 and LS-32 systems and were correctly differentiated based upon their respective melt profiles. Short STR amplicon melt curves were affected by repeat number, and single-source and mixed DNA samples were additionally differentiated by the formation of heteroduplexes. Melting curves were shown to be unique and reproducible from DNA quantities ranging from 20 to 0.4 ng and distinguished identical from nonidentical genotypes from DNA derived from different biological fluids and compromised samples. Thus, a method is described which can assess both the quantity and the possible probative value of samples without full genotyping.