Vascular Stem Cells Research Articles

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

Root-Knot and Cyst Nematodes Activate Procambium-Associated Genes in Arabidopsis Roots.

Developmental plasticity is one of the most striking features of plant morphogenesis, as plants are able to vary their shapes in response to environmental cues. Biotic or abiotic stimuli often promote organogenesis events in plants not observed under normal growth conditions. Root-knot nematodes (RKNs) are known to parasitize multiple species of rooting plants and to induce characteristic tissue expansion called galls or root-knots on the roots of their hosts by perturbing the plant cellular machinery. Galls contain giant cells (GCs) and neighboring cells, and the GCs are a source of nutrients for the parasitizing nematode. Highly active cell proliferation was observed in galls. However, the underlying mechanisms that regulate the symptoms triggered by the plant-nematode interaction have not yet been elucidated. In this study, we deciphered the molecular mechanism of gall formation with an in vitro infection assay system using RKN Meloidogyne incognita, and the model plant Arabidopsis thaliana. By taking advantages of this system, we performed next-generation sequencing-based transcriptome profiling, and found that the expression of procambium identity-associated genes were enriched during gall formation. Clustering analyses with artificial xylogenic systems, together with the results of expression analyses of the candidate genes, showed a significant correlation between the induction of gall cells and procambium-associated cells. Furthermore, the promoters of several procambial marker genes such as ATHB8, TDR and WOX4 were activated not only in M. incognita-induced galls, but similarly in M. javanica induced-galls and Heterodera schachtii-induced syncytia. Our findings suggest that phytoparasitic nematodes modulate the host’s developmental regulation of the vascular stem cells during gall formation.

Abstract 4332: Interactome between vascular endothelial cells and Proneural glioma stem cells protects seeds for GBM recurrence from radiation therapy

Abstract Glioblastoma (GBM) is the most aggressive form of intracranial tumors. Despite multimodal treatment strategies that include surgical resection, radiation and chemotherapy, most patients survive for less than two years. GBM cells tend to invade into adjacent normal brain tissues and therefore, cannot be completely resected by surgery. These remaining tumor cells are the “seeds” to escape post-surgical therapies, thereby giving rise to recurrent tumors during or after treatment. The exact mechanisms by which these seeds contain pre-existing resistant cells, and/or gain resistance to therapy remain elusive. Recent findings demonstrated that communication between normal and GBM cells play physiological roles in tumor initiation and progression. Here, we aim to determine how vascular endothelial (VE) cells affect GBM recurrence in response to radiotherapy. To this end, we first assessed the effect of secreted factors from VE cells on patient-derived GBM neurospheres in vitro by using their conditioned medium (CM). GBM neurospheres promoted their growth with CM from VE cells. qPCR and FACS exhibited that the CM treatment preferentially protected CD133+ population in GBM neurospheres that otherwise were depleted by irradiation treatment. Furthermore, RNA sequencing of VE cells and A2B5-expressing GBM stem-like cells directly isolated from GBM and epilepsy patients demonstrated that only tumor-derived VE cells and GBM stem-like cells express a high level of a secreted factor Endocan (ESM1) and its receptor CD11a, respectively. Preferential expression of CD11a in perivascular GBM cells was also confirmed by immunocytochemistry. Further, we found that recombinant Endocan is able to promote GBM neurosphere growth in vitro after irradiation and protected them from irradiation-induced aggressive mesenchymal transition of these cells. Taken together, these results demonstrate a potential role of a crosstalk between peri-vascular GBM stem-like cells and their adjacent VE cells, via the ligand-receptor interaction mechanism. This interactome network may help Proneural GBM stem cells to escape from radiation therapy, thereby reinitiating the tumor as recurrence via an Endocan-CD11a signaling. Hence, targeting Endocan in endothelial cells or CD11a in GBM cells may have potential as a novel efficacious strategy for preventing recurrence. Citation Format: Soniya Bastola, Marat S. Pavlyukov, Shinobu Yamaguchi, Jun Wang, Svetlana Komarova, Harley I. kornblum, Ichiro Nakano. Interactome between vascular endothelial cells and Proneural glioma stem cells protects seeds for GBM recurrence from radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4332. doi:10.1158/1538-7445.AM2017-4332

Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion.

The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors, and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promoted endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels show superior sensitivity and reproducibility over Matrigel when evaluating known inhibitors, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the US Environmental Protection Agency. The identified synthetic hydrogels should be suitable alternatives to Matrigel for common cell-culture applications.

InVitro Generation of Vascular Wall-Resident Multipotent Stem Cells ofMesenchymal Nature from Murine Induced Pluripotent Stem Cells.

SummaryThe vascular wall (VW) serves as a niche for mesenchymal stem cells (MSCs). In general, tissue-specific stem cells differentiate mainly to the tissue type from which they derive, indicating that there is a certain code or priming within the cells as determined by the tissue of origin. Here we report the in vitro generation of VW-typical MSCs from induced pluripotent stem cells (iPSCs), based on a VW-MSC-specific gene code. Using a lentiviral vector expressing the so-called Yamanaka factors, we reprogrammed tail dermal fibroblasts from transgenic mice containing the GFP gene integrated into the Nestin-locus (NEST-iPSCs) to facilitate lineage tracing after subsequent MSC differentiation. A lentiviral vector expressing a small set of recently identified human VW-MSC-specific HOX genes then induced MSC differentiation. This direct programming approach successfully mediated the generation of VW-typical MSCs with classical MSC characteristics, both in vitro and in vivo.

Identification of cyst nematode B-type CLE peptides and modulation of the vascular stem cell pathway for feeding cell formation.

Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular procambium/cambium are regulated by CLE-receptor kinase-WOX signaling modules. Previous data showed that cyst nematode CLE-like effector proteins delivered into host cells through a stylet, act as ligand mimics of plant A-type CLE peptides and are pivotal for successful parasitism. Here we report the identification of a new class of CLE peptides from cyst nematodes with functional similarity to the B-type CLE peptide TDIF (tracheary element differentiation inhibitory factor) encoded by the CLE41 and CLE44 genes in Arabidopsis. We further demonstrate that the TDIF-TDR (TDIF receptor)-WOX4 pathway, which promotes procambial meristem cell proliferation, is involved in beet cyst nematode Heterodera schachtii parasitism. We observed activation of the TDIF pathway in developing feeding sites, reduced nematode infection in cle41 and tdr-1 wox4-1 mutants, and compromised syncytium size in cle41, tdr-1, wox4-1 and tdr-1 wox4-1 mutants. By qRT-PCR and promoter:GUS analyses, we showed that the expression of WOX4 is decreased in a clv1-101 clv2-101 rpk2-5 mutant, suggesting that WOX4 is a potential downstream target of nematode CLEs. Exogenous treatment with both nematode A-type and B-type CLE peptides induced massive cell proliferation in wild type roots, suggesting that the two types of CLEs may regulate cell proliferation during feeding site formation. These findings highlight an important role of the procambial cell proliferation pathway in cyst nematode feeding site formation.

Stem Cells for Diabetes Complications: A Future Potential Cure.

Long-standing diabetes leads to structural and functional alterations in both the micro- and the macrovasculature. Designing therapies to repair these abnormalities present unique and sophisticated challenges. Vascular endothelial cells are the primary cells damaged by hyperglycemia-induced adverse effects. Vascular stem cells that give rise to endothelial progenitor cells and mesenchymal progenitor cells represent an attractive target for cell therapy for diabetic patients. In this review, we shed light on challenges and recent advances surrounding stem cell therapies for diabetes vascular complications and discuss limitations for their clinical adoption.

Contribution of Vascular Cells to Neointimal Formation

The de-differentiation and proliferation of smooth muscle cells (SMCs) are widely accepted as the major contributor to vascular remodeling. However, recent studies indicate that vascular stem cells (VSCs) also play an important role, but their relative contribution remains to be elucidated. In this study, we used genetic lineage tracing approach to further investigate the contribution of SMCs and VSCs to neointimal thickening in response to endothelium denudation injury or artery ligation. In vitro and in vivo analysis of MYH11-cre/Rosa-loxP-RFP mouse artery showed that SMCs proliferated at a much slower rate than non-SMCs. Upon denudation or ligation injury, two distinct types of neointima were identified: Type-I neointimal cells mainly involved SMCs, while Type II mainly involved non-SMCs. Using Sox10-cre/Rosa-loxP-LacZ mice, we found that Sox10+ cells were one of the cell sources in neointima. In addition, lineage tracing using Tie2-cre/Rosa-LoxP-RFP showed that endothelial cells also contributed to the neointimal formation, but rarely transdifferentiated into mesenchymal lineages. These results provide a novel insight into the contribution of vascular cells to neointima formation, and have significant impact on the development of more effective therapies that target specific vascular cell types.

Vascular Endothelial Growth Factor Up-regulation in Human Amniotic Fluid Stem Cell Enhances Nephroprotection After Ischemia-Reperfusion Injury in the Rat.

To evaluate if the up-regulation of vascular endothelial growth factor strengthens the protective effect of amniotic fluid stem cells in a renal ischemia-reperfusion injury model. Randomized animal study. University research laboratory. A total of 40 males 12-week-old Wistar rats were subjected to ischemia-reperfusion and assigned to four groups: amniotic fluid stem cells, vascular endothelial growth factor-amniotic fluid stem cells in two different doses, and vehicle. Ten animals were used as sham-controls. Six hours after induction of renal ischemia-reperfusion injury, amniotic fluid stem cells, vascular endothelial growth factor-amniotic fluid stem cells in two different doses, or vehicle were injected intraarterially. Analyses were performed at 24 hours, 48 hours, and 2 months after treatment. Outcome measures included serum creatinine, urine microprotenuira, and immunohistomorphometric analyses. Vascular endothelial growth factor-amniotic fluid stem cells induced a significantly higher nephroprotection than amniotic fluid stem cells. This effect was mediated mainly by immunomodulation, which led to lower macrophage infiltration and higher presence of regulatory T cell after ischemia-reperfusion injury. At medium term, it inhibited the progression toward chronic kidney disease. Vascular endothelial growth factor-amniotic fluid stem cells can worsen the ischemia-reperfusion injury when delivered in a high dose. Up-regulation of vascular endothelial growth factor enhances the therapeutic effect of human amniotic fluid stem cells in rats with renal ischemia-reperfusion injury, mainly by mitogenic, angiogenic, and anti-inflammatory mechanisms.

Functional Role of Cardiovascular Exosomes in Myocardial Injury and Atherosclerosis.

Extracellular vesicles are now widely recognized as key players in the prevention, repair or progression of cardiovascular disease. Here we first focus on the functional roles of extracellular vesicles in the cross-talk between cardiomyocytes and endothelial cells, important for maintaining normal development and function of the heart. Second, we discuss the role of extracellular vesicles secreted by embryonic and non-embryonic stem cells in repairing cardiomyocyte function and in restoring angiogenic potential after myocardial ischemia-reperfusion injury. Third, we focus on the role of extracellular vesicles in Endothelial to Mesenchymal Transition (EndMT), leading to conversion of endothelial cells to fibroblasts, secretion of extracellular proteins collagen and fibronectin, and fibrosis. Finally, we discuss the role of extracellular vesicles secreted under stress by endothelial cells, macrophages and vascular smooth muscle cells in atherosclerosis. On aggregate, the reviewed preclinical studies present evidence that extracellular vesicles secreted by cardiomyocytes, fibroblasts, endothelial cells, immune-system-related cells, vascular smooth muscle cells and stem cells play an important role in the pathogenesis of cardiovascular disease. However, further studies are needed to gain better insight into the mechanisms used to select specific content to transfer to specific target cells, and to induce or repress signaling pathways in their target cells.

Generation of Antigen-Presenting Cells from Human Vascular Adventitia-Resident Progenitors

Macrophages and dendritic cells play a central role in the immune system. Here, we identify CD44(+) vascular wall-resident multipotent stem cells (VW-MPSCs) as progenitors for macrophages and dendritic cell-like cells. Using human internal thoracic artery (hITA) tissue specimens we performed ex vivo sprouting assays followed by immunohistochemistry. hITA rings and hITA wall-derived cells were treated with GM-CSF+IL-4. The cells were then characterized by immunostaining and flow cytometry for CD31, CD34, CD44, CD45, CD68 and CD80. While neither CD68(+) nor CD80(+) cells were found in freshly isolated hITA sections, they were present after sprouting assay. Double immunofluorescence analysis after sprouting assay revealed the presence of both CD45(+)CD68(+) and C44(+)CD68(+) macrophages as well as C44(+)CD80(+) dendritic cell-like cells within the adventitia and among the sprouting cells.

A New Catheter Technology to Deliver Vascular Stem-Cells

A new device has been designed, developed and tested to improve the capacity of vascular drug and stem cell delivery. The device consists of a catheter with a multitude of small lumens (instead of a large central channel lumen).

Neuropeptide Y Is Critical for Bone Marrow Stromal Cells and Hematopoietic Recovery Following Injury

Bone marrow suppression is the most common limiting side-effect of conventional cancer chemo/radio therapy and is the primary cause of morbidity/mortality after accidental exposure to a high dose of ionizing radiation. The mechanisms mediating radiation-induced hematopoietic stem and stromal cell dysfunction however are not well understood. Radiation therapy causes substantial sensory neuropathy in patients. Recent studies reveal that bone marrow cells are highly innervated by sympathetic nerve fibers and that chemotherapy induced nerve-damage can impair hematopoietic regeneration, suggesting a contribution of nerve fibers in the regulation of hematopoietic stem cell and stromal cell activities. Whether irradiation- mediated nerve injury is a crucial lesion that causes deficits in hematopoietic recovery is not known. We recently discovered that differential signaling from the neuropeptide Y (NPY) receptors on bone marrow endothelial cells regulates vascular permeability and stem cell egress. NPY is an important neurotransmitter of the sympathetic nervous system and the principal adreno-medullary hormone. In this study, we found that NPY is important for reconstitution of the bone marrow niche and hematopoietic regeneration following sublethal irradiation (650 cGy). The levels of NPY were significantly reduced in bone marrow of irradiated mice suggesting damage to nerve fibers. Treatment of wild-type mice with full length NPY (1µg/mouse/day) for 3 consecutive days after irradiation markedly reduced the loss of mesenchymal stem cells (CD45-Ter119-CD31-Nestin+PDGFR+CD51+), endothelial cells (CD45-Ter119-CD31+VE-cadherin+) and hematopoietic stem and progenitor cells (SLAM LSK and LSK) in the bone marrow and promoted faster hematopoietic recovery. In addition, pharmacological NPY treatment prevented irradiation mediated nerve fiber damage. In contrast, in NPY knockout mice, regeneration of CD45neg stromal cells, SLAM LSK and LSK cells after irradiation was significantly reduced compared to wild-type controls. This reduced hematopoietic recovery in NPY deficient mice following irradiation was associated with increased apoptosis/necrosis of stromal cells and hematopoietic stem and progenitor cells. We also examined whether NPY played an intrinsic or extrinsic role in stem cell homing. Wild-type or NPY deficient BM cells were transplanted into wild-type or NPY knockout recipients. Strikingly, the homing of wild-type donor cells into NPY deficient recipients or NPY knockout donor cells into wild-type recipients were both reduced. To explore whether NPY regulates human stem cells, we treated human cord blood CD34+ cells ex vivo with NPY for 3 days and evaluated cell expansion. Long-term culture assays demonstrated that NPY treatment enhanced the clonal expansion of CD34+ cells. In conclusion, our studies suggests that NPY plays both an intrinsic and extrinsic role in hematopoiesis and that NPY-mediated protection of the sympathetic nervous system within the bone marrow can facilitate stem cell niche regeneration and enhance regenerative hematopoiesis following irradiation/injury. DisclosuresNo relevant conflicts of interest to declare.

ERECTA-family receptor kinase genes redundantly prevent premature progression of secondary growth in the Arabidopsis hypocotyl.

Secondary growth is driven by continuous cell proliferation and differentiation of the cambium that acts as vascular stem cells, producing xylem and phloem to expand vascular tissues laterally. During secondary growth of hypocotyls in Arabidopsis thaliana, the xylem undergoes a drastic phase transition from a parenchyma-producing phase to a fiber-producing phase at the appropriate time. However, it remains to be fully elucidated how progression of secondary growth is properly controlled. We focused on phenotypes of hypocotyl vasculatures caused by double mutation in ERECTA (ER) and ER-LIKE1 (ERL1) receptor-kinase genes to elucidate their roles in secondary growth. ER and ERL1 redundantly suppressed excessive radial growth of the hypocotyl vasculature during secondary growth. ER and ERL1 also prevented premature initiation of the fiber differentiation process mediated by the NAC SECONDARY WALL THICKENING PROMOTING FACTORs in the hypocotyl xylem. Upon floral transition, the hypocotyl xylem gained a competency to respond to GA in a BREVIPEDICELLUS-dependent manner, which was a prerequisite for fiber differentiation. However, even after the floral transition, ER and ERL1 prevented precocious initiation of the GA-mediated fiber formation. Collectively, our findings reveal that ER and ERL1 redundantly prevent premature progression of sequential events in secondary growth.

Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by Klf4.

The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (adventitial sca1-positive progenitor cells [AdvSca1]), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo. Although emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and heterogeneity, and contribution to vessel remodeling. Using 2 in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. After vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo, whereas in vitro approaches demonstrate that Klf4 is essential for the maintenance of the AdvSca1 progenitor phenotype. We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

Regulation of Hematopoiesis and Osteogenesis by Blood Vessel-Derived Signals.

In addition to their conventional role as a versatile transport system, blood vessels provide signals controlling organ development, regeneration, and stem cell behavior. In the skeletal system, certain capillaries support perivascular osteoprogenitor cells and thereby control bone formation. Blood vessels are also a critical component of niche microenvironments for hematopoietic stem cells. Here we discuss key pathways and factors controlling endothelial cell behavior in bone, the role of vessels in osteogenesis, and the nature of vascular stem cell niches in bone marrow.

The vascular adventitia: An endogenous, omnipresent source of stem cells in the body.

Until a decade ago it was believed that the wall of adult blood vessels exclusively contains terminally differentiated cell types. A paradigm shift was unavoidable since studies from different groups convincingly showed the presence of vascular wall-resident stem and progenitor cells (VW-SCs) which were identified to particularly reside in the sub-endothelial space and the so-called adventitial "vasculogenic zone". Data published during the last decade uncloaked the fact that VW-SCs have the capacity to differentiate into both vascular and non-vascular cell types. Up to date, little is known about the full capacity of VW-SCs, the exact composition of their endogenous niche and the mechanisms that govern their self-renewal, activation and differentiation. The aim of this review is to provide an overview about the current knowledge on VW-SCs and to highlight the impact of this endogenous niche on health and disease. In addition, we will discuss strategies how these adult stem cells could be manipulated in order to activate and expand them, ideally within their niche at sites of tissue damage and subsequently differentiate them into a desired cell type, e.g. an endothelial cell, a macrophage or a muscle cell. This would pave the way towards new pharmacological strategies for endogenous tissue repair and regeneration.

Vascular Procr+ stem cells: Finding new branches while looking for the roots.

Generation and growth of the blood vasculature network is a highly synchronized process, requiring coordinated efforts of endothelial cells and pericytes to maintain blood vessel integrity and regeneration. In a recent paper published in Cell Research, Yu et al. identified and characterized bipotent Procr-expressing vascular endothelial stem cells, which give rise to both endothelial cells and pericytes.

Identification of blood vascular endothelial stem cells by the expression of protein C receptor.

Vascular growth and remodeling are dependent on the generation of new endothelial cells from stem cells and the involvement of perivascular cells to maintain vessel integrity and function. The existence and cellular identity of vascular endothelial stem cells (VESCs) remain unclear. The perivascular pericytes in adult tissues are thought to arise from the recruitment and differentiation of mesenchymal progenitors during early development. In this study, we identified Protein C receptor-expressing (Procr+) endothelial cells as VESCs in multiple tissues. Procr+ VESCs exhibit robust clonogenicity in culture, high vessel reconstitution efficiency in transplantation, long-term clonal expansion in lineage tracing, and EndMT characteristics. Moreover, Procr+ VESCs are bipotent, giving rise to de novo formation of endothelial cells and pericytes. This represents a novel origin of pericytes in adult angiogenesis, reshaping our understanding of blood vessel development and homeostatic process. Our study may also provide a more precise therapeutic target to inhibit pathological angiogenesis and tumor growth.

Vascular stem cells-potential for clinical application.

Cell therapy is a growing area of research as an alternative to pharmaceuticals or surgery for the treatment of ischaemic disease. Studies are focusing on delivering tissue-derived cells into damaged organs to promote vascular regeneration or gain of function. Pubmed, clinicaltrials.gov, BHF website. Stem cells have the potential to become a viable treatment for many diseases, as indicated by the numerous pre-clinical studies demonstrating therapeutic benefit. The mechanisms of action for transplanted stem cells are still open to debate. Proposed mechanism includes direct cell incorporation and paracrine action. Additionally, the secretome produced by transplanted cells remains largely unknown. Initial studies focused on delivering stem cells by injection; however, current research is utilizing biomaterials to target cell delivery to specific areas. Whilst stem cell research in the laboratory is expanding rapidly, transition into clinical studies is hindered by the availability of equivalent clinical grade reagents.