Mesenchymal stem/stromal cells (MSCs) are distributed in various tissues and are used in clinical applications as a source of transplanted cells because of their easy harvestability. Although MSCs express numerous cell-surface antigens, single-cell analyses have revealed a highly heterogeneous cell population depending on the original tissue and donor conditions, including age and interindividual differences. This heterogeneity leads to differences in their functions, such as multipotency and immunomodulatory effects, making it challenging to effectively treat targeted diseases. The therapeutic efficacy of MSCs is controversial and depends on the implantation site. Thus, there is no established recipe for the transplantation of MSCs (including the type of disease, type of origin, method of cell culture, form of transplanted cells, and site of delivery). Our recent preclinical study identified appropriate MSCs and their suitable transplantation routes in a mouse model of inflammatory bowel disease (IBD). Three-dimensional (3D) cultures of MSCs have been demonstrated to enhance their properties and sustain engraftment at the lesion site. In this note, we explore the methods of MSC transplantation for treating IBDs, especially Crohn\'s disease, from clinical trials published over the past decade. Given the functional changes in MSCs in 3D culture, we also investigate the clinical trials using 3D constructs of MSCs and explore suitable diseases that might benefit from this approach. Furthermore, we discuss the advantages of the prospective isolation of MSCs in terms of interindividual variability. This note highlights the need to define the method of MSC transplantation, including interindividual variability, the culture period, and the transplantation route.

Cell surface marker expression is one of the criteria for defining human mesenchymal stem or stromal cells (MSC) in vitro. However, it is unclear if expression of markers including CD73 and CD90 reflects the in vivo origin of cultured cells. We evaluated expression of 15 putative MSC markers in primary cultured cells from periosteum and cartilage to determine whether expression of these markers reflects either the differentiation state of cultured cells or the self-renewal of in vivo populations. Cultured cells had universal and consistent expression of various putative stem cell markers including > 95% expression CD73, CD90 and PDPN in both periosteal and cartilage cultures. Altering the culture surface with extracellular matrix coatings had minimal effect on cell surface marker expression. Osteogenic differentiation led to loss of CD106 and CD146 expression, however CD73 and CD90 were retained in > 90% of cells. We sorted freshly isolated periosteal populations capable of CFU-F formation on the basis of CD90 expression in combination with CD34, CD73 and CD26. All primary cultures universally expressed CD73 and CD90 and lacked CD34, irrespective of the expression of these markers ex vivo indicating phenotypic convergence in vitro. We conclude that markers including CD73 and CD90 are acquired in vitro in most \'mesenchymal\' cells capable of expansion. Overall, we demonstrate that in vitro expression of many cell surface markers in plastic-adherent cultures is unrelated to their expression prior to culture.

The periosteum plays a crucial role in bone healing and is an important source of skeletal stem and progenitor cells. Recent studies in mice indicate that diverse populations of skeletal progenitors contribute to growth, homeostasis and healing. Information about the in vivo identity and diversity of skeletal stem and progenitor cells in different compartments of the adult human skeleton is limited. In this study, we compared non-hematopoietic populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-dimensional clustering analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+CD146- population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations in vitro. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. CD90+ cells are also highly enriched for CFU-F in bone marrow and endosteum, but not articular cartilage. In conclusion, our study indicates considerable diversity in the non-hematopoietic cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal progenitor cells reside within the CD90+CD34+ population.

Pericytes are mural cells closely associated with endothelial cells in capillaries and microvessels. They are precursors of mesenchymal stem/stromal cells that have historically been retrospectively characterized in culture. We established a protocol, described in this chapter, to characterize and isolate pericytes from multiple human organs by flow cytometry and fluorescence-activated cell sorting. This prospective purification of pericytes brings us a step forward in the development of strategies for their use in the clinic.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

In this study, we show that matrix dense cortical bone is the more potent compartment of bone than bone marrow as a stromal source for mesenchymal stem cells as isolated from adult rats. Lineage-depleted cortical bone-mesenchymal stem cells demonstrated >150-fold enrichment of colony forming unit-fibroblasts per cell incidence. compared to lineage-depleted bone marrow-mesenchymal stem cells, corresponding to a 70-fold increase in absolute recovered colony forming unit-fibroblasts. The composite phenotype Lin(-)/CD45(-)/CD31(-)/VLA-1(+)/Thy-1(+) enriched for clonogenic mesenchymal stem cells solely from cortical bone-derived cells from which 70% of clones spontaneously differentiated into all lineages of bone, cartilage, and adipose. Both populations generated vascularized bone tissue within subcutaneous implanted collagen scaffolds; however, cortical bone-derived cells formed significantly more osteoid than bone marrow counterparts, quantified by histology. The data demonstrate that our isolation protocol identifies and validates mesenchymal stem cells with superior clonal, proliferative, and developmental potential from cortical bone compared to the bone marrow niche although marrow persists as the typical source for mesenchymal stem cells both in the literature and current pre-clinical therapies.

Cell-mediated therapy is currently considered as a novel approach for many human diseases. Potential uses range from topic applications with the regeneration of confined tissue areas to systemic applications. Stem cells including mesenchymal stroma/stem cells (MSCs) represent a highly attractive option. Their potential to cure or alleviate human diseases is investigated in a number of clinical trials. A wide variety of methods has been established in the past years for isolation, cultivation and characterization of human MSCs as expansion is presently deemed a prerequisite for clinical application with high numbers of cells carrying reproducible properties. MSCs have been retrieved from various tissues and used in a multitude of settings whereby numerous experimental protocols are available for expansion of MSCs in vitro. Accordingly, different isolation, culture and upscaling techniques contribute to the heterogeneity of MSC characteristics and the, sometimes, controversial results. Therefore, this review discusses and summarizes certain experimental conditions for MSC in vitro culture focusing on adult bone marrow-derived and neonatal umbilical cord-derived MSCs in order to enhance our understanding for MSC tissue sources and to stratify different procedures. Copyright © 2016 John Wiley & Sons, Ltd.

OBJECTIVE: Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective.
METHODS: We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus-specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immuno-magnetically isolating them via the transiently up-regulated activation marker, CD137.
RESULTS: In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From 2 × 10(9) peripheral blood mononuclear cells, we isolated 27 × 10(3)-318 × 10(3)Aspergillus-specific T-helper cells. Frequency among total T cells was increased, on average, by 200-fold. Specific studies indicate specificity and functionality: After non-specific in vitro expansion and re-stimulation with different antigens, we observed strong cytokine responses to A. fumigatus and some other fungi including Candida albicans, but none to unrelated antigens.
CONCLUSIONS: Our technology isolates naturally occurring Aspergillus-specific T-helper cells within 2 days of identifying the clinical indication. Rapid adoptive transfer of Aspergillus-specific T cells may be quite feasible; the clinical benefit remains to be demonstrated. A manufacturing license as an advanced-therapy medicinal product was received and a clinical trial in post-transplantation invasive aspergillosis patients approved. The product is dosed at 5 × 10E3/kg T cells (single intravenous injection), of which at least 10% must be A. fumigatus-specific.

OBJECTIVE: Traditionally, stem cell therapy for myocardial infarction (MI) has been administered as a single treatment in the acute or subacute period after MI. These time intervals coincide with marked differences in the post-infarct myocardial environment, raising the prospect that repeat cell dosing could provide incremental benefit beyond a solitary intervention. This prospect was evaluated with the use of mesenchymal stromal cells (MSCs).
METHODS: Three groups of rats were studied. Single-therapy and dual-therapy groups received allogeneic, prospectively isolated MSCs (1 × 10(6) cells) by trans-epicardial injection immediately after MI, with additional dosing 1 week later in the dual-therapy cohort. Control animals received cryopreservant solution only. Left ventricular (LV) dimensions and ejection fraction (EF) were assessed by cardiac magnetic resonance immediately before MI and at 1, 2 and 4 weeks after MI.
RESULTS: Immediate MSC treatment attenuated early myocardial damage with EF of 35.3 ± 3.1% (dual group, n = 12) and 35.2 ± 2.2% (single group, n = 15) at 1 week after MI compared with 22.1 ± 1.9% in controls (n = 17, P < 0.01). In animals receiving a second dose of MSCs, EF increased to 40.7 ± 3.1% by week 4, which was significantly higher than in the single-therapy group (EF 35.9 ± 1.8%, P < 0.05). Dual MSC treatment was also associated with greater myocardial mass and arteriolar density, with trends toward reduced myocardial fibrosis. These incremental benefits were especially observed in remote (non-infarct) segments of LV myocardium.
CONCLUSIONS: Repeated stem cell intervention in both the acute and the sub-acute period after MI provides additional improvement in ventricular function beyond solitary cell dosing, largely owing to beneficial changes remote to the area of infarction.