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The potential of obtaining cell cultures with neural crest resemblance (neural crest‐derived stem cells [NCSCs]) from dental‐related tissues, including human dental pulp cells (hDPCs), has been discussed in the literature. However, most reports include the use of serum‐rich conditions and do not describe the potential for neural differentiation, slowing translation to the clinic. Therefore, we aimed to culture and characterize NCSCs from the human dental pulp in vitro and evaluate their ability to differentiate into neurons; we also investigated the effectiveness of the addition of BMP4 to enhance this potential. Cultures were established from a varied cohort of patient samples and grown, as monolayers, in serum, serum‐free, and also under sphere‐aggregation conditions to induce and identify a NCSC phenotype. hDPC cultures were characterized by immunocytochemistry and reverse transcription quantitative polymerase chain reaction. Monolayer cultures expressed stem cell, neural progenitor and neural crest‐related markers. Culturing hDPCs as neurospheres (hDPC‐NCSCs) resulted in an increased expression of neural crest‐related genes, while the addition of BMP4 appeared to produce better NCSC characteristics and neural differentiation. The neural‐like phenotype was evidenced by the expression of TUJ1, peripherin, NFH, TAU, SYN1, and GAP43. Our results describe the establishment of hDPC cultures from a large variety of patients in serum‐free medium, as NCSC that differentiate into neural‐like cells, as well as an important effect of BMP4 in enhancing the neural crest phenotype and differentiation of hDPCs. Human dental pulp tissue collected from a varied cohort of samples was used to directly establish monolayer cultures in defined serum‐free conditions. In monolayer, the cultures maintained an mesenchymal stem cell (MSC) phenotype regardless of the initial serum‐free conditions. A Neural crest‐derived stem cell phenotype was favored by adding BMP4 before or after transferring the cells to a 3D culture.

Despite approaches in regenerative medicine using stem cells, bio‐engineered scaffolds, and targeted drug delivery to enhance human tissue repair, clinicians remain unable to regenerate large‐scale, multi‐tissue defects in situ. The study of regenerative biology using mammalian models of complex tissue regeneration offers an opportunity to discover key factors that stimulate a regenerative rather than fibrotic response to injury. For example, although primates and rodents can regenerate their distal digit tips, they heal more proximal amputations with scar tissue. Rabbits and African spiny mice re‐grow tissue to fill large musculoskeletal defects through their ear pinna, while other mammals fail to regenerate identical defects and instead heal ear holes through fibrotic repair. This Review explores the utility of these comparative healing models using the spiny mouse ear pinna and the mouse digit tip to consider how mechanistic insight into reparative regeneration might serve to advance regenerative medicine. Specifically, we consider how inflammation and immunity, extracellular matrix composition, and controlled cell proliferation intersect to establish a pro‐regenerative microenvironment in response to injuries. Understanding how some mammals naturally regenerate complex tissue can provide a blueprint for how we might manipulate the injury microenvironment to enhance regenerative abilities in humans. Stem Cells Translational Medicine 2018;7:220–231 Comparative models of regenerating and non‐regenerating mammals (i.e., ear pinna regeneration in Acomys vs. Mus) will help uncover key molecular and cellular differences underlying a regenerative response to injury. Importantly, a comparative approach will shed light on how the injury microenvironment stimulates blastema formation in lieu of fibrotic repair.

Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell‐based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell‐derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell‐derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibrosis. Exosomes were isolated and evaluated for their protein and miRNA cargo. Direct effects of hAEC Exo on immune cell function, including macrophage polarization, phagocytosis, neutrophil myeloperoxidase activity and T cell proliferation and uptake, were measured. Their impact on immune response, histological outcomes, and bronchioalveolar stem cell (BASC) response was assessed in vivo following bleomycin challenge in young and aged mice. hAEC Exo carry protein cargo enriched for MAPK signaling pathways, apoptotic and developmental biology pathways and miRNA enriched for PI3K‐Akt, Ras, Hippo, TGFβ, and focal adhesion pathways. hAEC Exo polarized and increased macrophage phagocytosis, reduced neutrophil myeloperoxidases, and suppressed T cell proliferation directly. Intranasal instillation of 10 μg hAEC Exo 1 day following bleomycin challenge reduced lung inflammation, while treatment at day 7 improved tissue‐to‐airspace ratio and reduced fibrosis. Administration of hAEC Exo coincided with the proliferation of BASC. These effects were reproducible in bleomycin‐challenged aged mice. The paracrine effects of hAECs can be largely attributed to their exosomes and exploitation of hAEC Exo as a therapy for IPF should be explored further. Stem Cells Translational Medicine 2018;7:180–196 Human amnion epithelial cells (hAECs) possess potent reparative and regenerative properties, especially in experimental models of lung fibrosis. It is now evident that these mechanism may be attributed to secretory factors released by hAECs termed exosomes. These hAEC Exo can be isolated with high purity through differential ultracentrifugation. Transmission electron microscopy shows exosomes of typical cup‐shaped morphology, intracellular and outward budding vesicles from hAECs (Scale bar = 100 nm). Administration of 10 μg of hAEC Exo into bleomycin challenged mice ameliorated fibroblast activated αSMA positive cells (Scale bar = 50 μm) and increased lung progenitor cell renewal seen through expression of CC10+/SPC+ dual positive staining for bronchioalveolar stem cells (Scale bar = 50 μm).

In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous‐deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild‐type mouse LGs by transplanting them into the LGs of TSP ‐1−/− mice, which represent a novel mouse model for ADDE. TSP‐1−/− mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c‐kit‐positive epithelial cell adhesion molecule (EpCAM+) populations sorted from mouse LGs, the c‐kit+dim/EpCAM+/Sca1 − /CD34 − /CD45 − cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three‐dimensional cultures. Moreover, when transplanted into injured or “diseased” LGs, they engraft into acinar and ductal compartments. EPCP‐injected TSP‐1−/− LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88–98

Gene therapy, cell therapy, and tissue engineering have the potential to revolutionize the treatment of disease and injury. Attaining marketing authorization for such advanced therapy medicinal products (ATMPs) requires a rigorous scientific evaluation by the European Medicines Agency—authorization is only granted if the product can fulfil stringent requirements for quality, safety, and efficacy. However, many ATMPs are being provided to patients under alternative means, such as “hospital exemption” schemes. Holoclar (ex vivo expanded autologous human corneal epithelial cells containing stem cells), a novel treatment for eye burns, is one of the few ATMPs to have been granted marketing authorization and is the first containing stem cells. This review highlights the differences in standards between an authorized and unauthorized medicinal product, and specifically discusses how the manufacture of Holoclar had to be updated to achieve authorization. The result is that patients will have access to a therapy that is manufactured to high commercial standards, and is supported by robust clinical safety and efficacy data. Stem Cells Translational Medicine 2018;7:146–154 The role of clonogenic keratinocytes in generation and renewal of the corneal epithelium. The holoclone differentiation process from highly proliferative self‐renewing holoclones to transiently amplifying cells (meroclones and paraclones). A confocal microscopy image of holoclone stem cells is on the left showing high expression of ΔNp63α, an isoform of the p63 transcription factor.

Mesenchymal stromal cells (MSCs) are immunomodulatory and tissue homeostatic cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular stromal cells (PSCs) identified within several different organs share characteristics of bone marrow‐derived MSCs (BM‐MSCs). These PSCs may also possess tissue‐specific properties and play a role in local tissue homeostasis. We hypothesized that human kidney‐derived PSCs (hkPSCs) would elicit improved kidney repair in comparison with BM‐MSCs. Here we introduce a novel, clinical‐grade isolation method of hkPSCs from cadaveric kidneys by enriching for the perivascular marker, NG2. hkPSCs show strong transcriptional similarities to BM‐MSCs but also show organotypic expression signatures, including the HoxD10 and HoxD11 nephrogenic transcription factors. Comparable to BM‐MSCs, hkPSCs showed immunosuppressive potential and, when cocultured with endothelial cells, vascular plexus formation was supported, which was specifically in the hkPSCs accompanied by an increased NG2 expression. hkPSCs did not undergo myofibroblast transformation after exposure to transforming growth factor‐β, further corroborating their potential regulatory role in tissue homeostasis. This was further supported by the observation that hkPSCs induced accelerated repair in a tubular epithelial wound scratch assay, which was mediated through hepatocyte growth factor release. In vivo, in a neonatal kidney injection model, hkPSCs reintegrated and survived in the interstitial compartment, whereas BM‐MSCs did not show this potential. Moreover, hkPSCs gave protection against the development of acute kidney injury in vivo in a model of rhabdomyolysis‐mediated nephrotoxicity. Overall, this suggests a superior therapeutic potential for the use of hkPSCs and their secretome in the treatment of kidney diseases. Stem Cells Translational Medicine 2017;6:405–418

Perinatal brain injury can lead to significant neurological and cognitive deficits and currently no therapies can regenerate the damaged brain. Neural stem cells (NSCs) have the potential to engraft and regenerate damaged brain tissue. The aim of this systematic review was to evaluate the preclinical literature to determine whether NSC administration is more effective than controls in decreasing perinatal brain injury. Controlled interventional studies of NSC therapy using animal models of perinatal brain injury were identified using MEDLINE and Embase. Primary outcomes were brain infarct size, motor, and cognitive function. Data for meta‐analysis were synthesized and expressed as standardized mean difference (SMD) with 95% confidence intervals (CI), using a random effects model. We also reported secondary outcomes including NSC survival, migration, differentiation, and effect on neuroinflammation. Eighteen studies met inclusion criteria. NSC administration decreased infarct size (SMD 1.09; CI: 0.44, 1.74, P = .001; I2 = 74%) improved motor function measured via the impaired forelimb preference test (SMD 2.27; CI: 0.85, 3.69, P = .002; I2 = 86%) and the rotarod test (SMD 1.88; CI: 0.09, 3.67, P = .04; I2 = 95%). Additionally, NSCs improved cognitive function measured via the Morris water maze test (SMD of 2.41; CI: 1.16, 3.66, P = .0002; I2 = 81%). Preclinical evidence suggests that NSC therapy is promising for the treatment of perinatal brain injury. We have identified key knowledge gaps, including the lack of large animal studies and uncertainty regarding the necessity of immunosuppression for NSC transplantation in neonates. These knowledge gaps should be addressed before NSC treatment can effectively progress to clinical trial. This systematic review and meta‐analysis shows that neural stem cells are a promising treatment for perinatal brain injury. Key knowledge gaps have been identified and should be addressed before the commencement of clinical trials.

The injection of minimally expanded exogenous mesenchymal stromal cells (MSCs) into a mouse model of human age‐related osteoporosis led to long‐term engraftment and markedly increased bone formation. This led to improved bone quality and turnover and sustained microarchitectural competence, establishing proof of concept that MSC transplantation may be used to prevent or treat human age‐related osteoporosis. Age‐related osteoporosis is driven by defects in the tissue‐resident mesenchymal stromal cells (MSCs), a heterogeneous population of musculoskeletal progenitors that includes skeletal stem cells. MSC decline leads to reduced bone formation, causing loss of bone volume and the breakdown of bony microarchitecture crucial to trabecular strength. Furthermore, the low‐turnover state precipitated by MSC loss leads to low‐quality bone that is unable to perform remodeling‐mediated maintenance—replacing old damaged bone with new healthy tissue. Using minimally expanded exogenous MSCs injected systemically into a mouse model of human age‐related osteoporosis, we show long‐term engraftment and markedly increased bone formation. This led to improved bone quality and turnover and, importantly, sustained microarchitectural competence. These data establish proof of concept that MSC transplantation may be used to prevent or treat human age‐related osteoporosis. Significance This study shows that a single dose of minimally expanded mesenchymal stromal cells (MSCs) injected systemically into a mouse model of human age‐related osteoporosis display long‐term engraftment and prevent the decline in bone formation, bone quality, and microarchitectural competence. This work adds to a growing body of evidence suggesting that the decline of MSCs associated with age‐related osteoporosis is a major transformative event in the progression of the disease. Furthermore, it establishes proof of concept that MSC transplantation may be a viable therapeutic strategy to treat or prevent human age‐related osteoporosis.

The umbilical cord has become an increasingly used source of mesenchymal stromal cells for preclinical and, more recently, clinical studies. Despite the increased activity, several aspects of this cell population have been under‐appreciated. Key issues are that consensus on the anatomical structures within the cord is lacking, and potentially different populations are identified as arising from a single source. To help address these points, we propose a histologically based nomenclature for cord structures and provide an analysis of their developmental origins and composition. Methods of cell isolation from Wharton's jelly are discussed and the immunophenotypic and clonal characteristics of the cells are evaluated. The perivascular origin of the cells is also addressed. Finally, clinical trials with umbilical cord cells are briefly reviewed. Interpreting the outcomes of the many clinical studies that have been undertaken with mesenchymal stromal cells from different tissue sources has been challenging, for many reasons. It is, therefore, particularly important that as umbilical cord cells are increasingly deployed therapeutically, we strive to better understand the derivation and functional characteristics of the cells from this important tissue source. Stem Cells Translational Medicine 2017;6:1620–1630 The human umbilical cord comprises an outer amniotic epithelium surrounding the mucoid connective tissue, Wharton's Jelly, which supports the three umbilical vessels. Wharton's Jelly can be divided into the perivascular compartment, which serves as the tunica adventia of the vessels (and contains the majority of mesenchymal stromal cells), and an intermediate zone that separates the perivascular compartment from the cord lining. Scale Bar = 5 mm.

Mesenchymal stem cells (MSCs) can show trisomy 7; however, the safety of these cells has not been fully investigated. The purposes of this study were to determine the ratio of patients whose synovial MSCs were transplanted clinically, to intensively investigate MSCs with trisomy 7 from a safety perspective, and to follow up the patients for 5 years after transplantation. Synovial MSCs at passage 0 were transplanted into a knee for degenerative meniscus tears in 10 patients, and the patients were checked at 5 years. The synovial MSCs were evaluated at passages 0 to 15 by G‐bands and digital karyotyping, and trisomy 7 was found in 3 of 10 patients. In those three patients, 5% to 10% of the synovial MSCs showed trisomy 7. The mRNA expressions of representative oncogenes and genes on chromosome 7 did not differ between MSCs with and without trisomy 7. Whole‐genome sequencing and DNA methylation analysis showed similar results for MSCs with and without trisomy 7. Transplantation of human synovial MSCs with trisomy 7 into eight mouse knees did not result in tumor formation under the skin or in the knees after 8 weeks in any mouse, whereas transplanted HT1080 cells formed tumors. In vitro chondrogenic potentials were similar between MSCs with and without trisomy 7. Five‐year follow‐ups revealed no serious adverse events in all 10 human patients, including 3 who had received MSCs with trisomy 7. Overall, our findings indicated that synovial MSCs with trisomy 7 were comparable with MSCs without trisomy 7 from a safety perspective. Trisomy 7 is often found in synovial mesenchymal stem cells (MSCs). However, the safety of these cells after transplantation has not been investigated. The authors found no serious adverse events, including tumor formation, in any of our 10 patients at 5 years after transplantation of MSCs with or without trisomy 7.