GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes.

It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.

Evaluation of the Therapeutic Potential of Human iPSCs in a Murine Model of VML.

Volumetric muscle loss injury is a common health problem with long-term disabilities. One common treatment is using muscle flaps from donor site, which has limited potentials due to donor site availability and morbidity. Although several stem cell therapies have been evaluated so far, most suffer from limited availability, immune incompatibility, or differentiation potential. Therefore, induced pluripotent stem cells (iPSCs) have a great promise for this purpose due to their unique differentiation, self-renewal, and immunocompatibility. Current study was designed to determine therapeutic potential of human iPSCs (hiPSCs) in a mouse model of volumetric muscle loss. Muscles were subjected to excision to generate 30%-40% muscle loss. Next, hiPSCs were differentiated toward skeletal myogenic progenitors and used with fibrin hydrogel to reconstruct the lost muscle. Histologic evaluation of the treated muscles indicated abundant engraftment of donor-derived mature fibers expressing human markers. Donor-derived fibers were also positive for the presence of neuromuscular junction (NMJ), indicating their proper innervation. Evaluation of the engrafted region indicated the presence of donor-derived satellite cells expressing human markers and Pax7. Finally, in situ muscle function analysis demonstrated significant improvement of the muscle contractility in muscles treated with hiPSCs. These results therefore provide key evidence for the therapeutic potential of human iPSCs in volumetric muscle loss injuries.

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.

Human pluripotent stem cell-derived chondroprogenitors for cartilage tissue engineering.

The cartilage of joints, such as meniscus and articular cartilage, is normally long lasting (i.e., permanent). However, once damaged, especially in large animals and humans, joint cartilage is not spontaneously repaired. Compensating the lack of repair activity by supplying cartilage-(re)forming cells, such as chondrocytes or mesenchymal stromal cells, or by transplanting a piece of normal cartilage, has been the basis of therapy for biological restoration of damaged joint cartilage. Unfortunately, current biological therapies face problems on a number of fronts. The joint cartilage is generated de novo from a specialized cell type, termed a 'joint progenitor' or 'interzone cell' during embryogenesis. Therefore, embryonic chondroprogenitors that mimic the property of joint progenitors might be the best type of cell for regenerating joint cartilage in the adult. Pluripotent stem cells (PSCs) are expected to differentiate in culture into any somatic cell type through processes that mimic embryogenesis, making human (h)PSCs a promising source of embryonic chondroprogenitors. The major research goals toward the clinical application of PSCs in joint cartilage regeneration are to (1) efficiently generate lineage-specific chondroprogenitors from hPSCs, (2) expand the chondroprogenitors to the number needed for therapy without loss of their chondrogenic activity, and (3) direct the in vivo or in vitro differentiation of the chondroprogenitors to articular or meniscal (i.e., permanent) chondrocytes rather than growth plate (i.e., transient) chondrocytes. This review is aimed at providing the current state of research toward meeting these goals. We also include our recent achievement of successful generation of "permanent-like" cartilage from long-term expandable, hPSC-derived ectomesenchymal chondroprogenitors.

A Myogenic Double-Reporter Human Pluripotent Stem Cell Line Allows Prospective Isolation of Skeletal Muscle Progenitors.

Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy.

Pre-transplantational Control of the Post-transplantational Fate of Human Pluripotent Stem Cell-Derived Cartilage.

Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Chondrogenic mesoderm from hPSCs and dedifferentiated nasal chondrocytes responded similarly to forskolin. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unmineralized chondrocytes after subcutaneous transplantation. Thus, the post-transplantational fate of chondrocytes originating from hPSC-derived chondroprogenitors can be controlled during their genesis in vitro.

Volumetric muscle loss injury repair using in situ fibrin gel cast seeded with muscle-derived stem cells (MDSCs).

Volumetric muscle defect, caused by trauma or combat injuries, is a major health concern leading to severe morbidity. It is characterized by partial or full thickness loss of muscle and its bio-scaffold, resulting in extensive fibrosis and scar formation. Therefore, the ideal therapeutic option is to use stem cells combined with bio-scaffolds to restore muscle. For this purpose, muscle-derived stem cells (MDSCs) are a great candidate due to their unique multi-lineage differentiation potential. In this study, we evaluated the regeneration potential of MDSCs for muscle loss repair using a novel in situ fibrin gel casting. Muscle defect was created by a partial thickness wedge resection in the tibialis anterior (TA) muscles of NSG mice which created an average of 25% mass loss. If untreated, this defect leads to severe muscle fibrosis. Next, MDSCs were delivered using a novel in situ fibrin gel casting method. Our results demonstrated MDSCs are able to engraft and form new myofibers in the defect when casted along with fibrin gel. LacZ labeled MDSCs were able to differentiate efficiently into new myofibers and significantly increase muscle mass. This was also accompanied by significant reduction of fibrotic tissue in the engrafted muscles. Furthermore, transplanted cells also contributed to new vessel formation and satellite cell seeding. These results confirmed the therapeutic potential of MDSCs and feasibility of direct in situ casting of fibrin/MDSC mixture to repair muscle mass defects.

Generation of an induced pluripotent stem cell line (CSCRMi001-A) from a patient with a new type of limb-girdle muscular dystrophy (LGMD) due to a missense mutation in POGLUT1 (Rumi).

Recently, a new type of limb-girdle muscular dystrophy (LGMD type 2Z) has been identified due to a missense mutation in POGLUT1 (protein O-glucosyltransferase-Rumi), an enzyme capable of adding glucose to a distinct serine residue of epidermal growth factor-like repeats containing a C-X-S-X-(P/A)-C consensus sequence such as Notch receptors. Affected patients demonstrate reduced Notch signaling, decreased muscle stem cell pool and hypoglycosylation of α-dystroglycan, leading to LGMD phenotype. Here we report the generation and characterization of an iPSC line (CSCRMi001-A) from a LGMD-2Z patient with missense mutation in POGLUT1 which can be used for in vitro disease modeling.

A unique microenvironment in the developing liver supports the expansion of megakaryocyte progenitors.

The fetal liver is the site of a major expansion of the hematopoietic stem cell (HSC) pool and is also a privileged organ to study megakaryocyte progenitor differentiation. We identified in the mouse fetal liver at day 13.5 a discrete stromal cell population harboring a CD45-TER119-CD31-CD51+VCAM-1+PDGFRα- (V+P-) phenotype that lacked colony-forming unit fibroblast activity and harbored an hepatocyte progenitor signature. This previously undescribed V+P- population efficiently supported megakaryocyte production from mouse bone marrow HSC and human peripheral blood HSC-myeloid progenitors cultured in the presence of limited cytokine concentrations. Megakaryocytes obtained in V+P- cocultures were polyploid, positive for CD41/CD42c, and efficiently produced proplatelets. Megakaryocyte production appeared to be mediated by an expansion of the progenitor compartment through HSC-stromal cell contact. In conclusion, the fetal liver contains a unique cellular microenvironment that could represent a platform for the discovery of regulators of megakaryopoiesis.

Skeletal muscle perfusion and stem cell delivery in muscle disorders using intra-femoral artery canulation in mice.

Muscular dystrophies are among major inherited muscle disorders characterized by progressive muscle damage and fibrosis with no definitive cure. Recently, gene or cell based therapies have been developed to restore the missing gene expression or replace the damaged tissues. In order to test the efficiency of these therapies in mice models of muscular dystrophies, the arterial route of delivery is very advantageous as it provides uniform muscle exposure to the therapeutic agents or cells. Although there are few reports of arterial delivery of the therapeutic agents or cells in mice, there is no in-depth description and evaluation of its efficacy in perfusion of downstream muscles. This study is aimed to develop a practical method for intra-femoral artery perfusion in mice and to evaluate perfusion efficiency using near-infrared-fluorescence (NIRF) imaging as well as histology following stem cell delivery. Our results provide a practical guide to perform this delicate method in mice. By using a sensitive fluorescent dye, different muscle groups of the hindlimb have been evaluated for proper perfusion. As the final step, we have validated the efficiency of arterial cell delivery into muscles using human iPS-derived myogenic cells in an immunodeficient mouse model for Duchenne muscular dystrophy (NSG-mdx(4cv)).

Long-term expandable SOX9+ chondrogenic ectomesenchymal cells from human pluripotent stem cells.

Here we report the successful generation and long-term expansion of SOX9-expressing CD271(+)PDGFRα(+)CD73(+) chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1(-)CD271(hi)PDGFRα(lo)CD73(-) neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming growth factorß (TGFß) inhibitor and fibroblast growth factor (FGF). When "primed" with TGFß, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B), cranial mesenchyme (ALX1/3/4), and chondroprogenitors (SOX9, COL2A1) of neural crest origin (SOX8/9, NGFR, NES). Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

Local cryoanalgesia is effective for tail-tip biopsy in mice.

Tail-tip biopsy for genotyping of genetically modified mice older than 21 d typically is performed by using isoflurane anesthesia. Isoflurane-induced changes in behavior and metabolism can result in unexpected complications and death. We investigated whether cryoanalgesia by using ethylene chloride spray would be an effective local anesthetic for tail-tip biopsies in mice. C57BL/6J mice were allocated randomly into 4 groups (n = 10 each) to receive isoflurane anesthesia with tail biopsy, ethylene chloride spray on the tip of the tail before biopsy, ethylene chloride spray without biopsy, or no treatment. Blood glucose was measured periodically in both groups undergoing tail biopsy, and the tail-pinch assay was performed in all mice that received ethylene chloride spray. Body weight, water, and food intake were measured daily for 2 wk. In both groups undergoing tail biopsy, blood glucose levels at 15 min were significantly higher than those after 2 min. This elevation was greater and more prolonged after 30 min in mice that received isoflurane compared with ethylene chloride spray. Tail-pinch latency at 20 min was greater than that after 2 min in all mice that received ethylene chloride spray. All mice gained weight, and there was no difference in food and water intake among groups. We conclude that ethylene chloride spray is an effective local anesthetic and a valuable alternative to isoflurane.

Multiple cystic sweat gland tumors in transgenic mice.

Here we describe gross and microscopic sweat gland tumors found in a transgenic mouse model of breast cancer, which had transforming growth factor α under the control of mouse mammary tumor virus promoter (MMTV-TGFα). Initially, 20% of the mice in the colony were affected. Cystic lesions formed on the phalanges, palmar surfaces of the metacarpals, and plantar surfaces of the metatarsals. The lesions were multifocal and nonulcerated with straw-colored fluid, ranging in size from 1 to 30 mm at the largest dimension. The colony was monitored for 6 mo; during that time, the prevalence of lesions increased to 52% of the mice. Histologically, in most cases the cyst walls were lined by 1 or 2 layers of normal-appearing epithelial cells that resembled basal cells, indicating adenoma. However, 2 cysts from 2 different mice had papillary proliferative projections and extensive disorganized glandular structures that protruded into the cyst cavities, indicating adenocarcinoma. In these 2 cases, the neoplastic cells revealed architectural and cytologic atypia with rare mitoses. Similar findings have previously been observed in sweat gland tumors; however, multiple sweat-gland tumors have not been reported in mice.

Effect of freshly isolated autologous tissue resident stromal cells on cardiac function and perfusion following acute myocardial infarction.

BACKGROUND: The aim of this study was to investigate the effect of intracoronary administration of freshly isolated, uncultured autologous tissue-derived stromal cells on cardiac function and perfusion after acute infarction in pigs. METHODS: A transmural myocardial infarction in a porcine model was induced by occlusion of the mid LAD with an angioplasty balloon for 3 h. Upon reperfusion, freshly isolated, uncultured autologous stromal cells (1.5×106 cells/kg) or control solution was injected into the infarct artery. Cardiac function and area at risk were determined by (99m)Tc-SPECT. RESULTS: Eight weeks after infarction, cell treated pigs showed a 20% smaller myocardial perfusion defect compared to control animals (35±9% vs. 44±5% of LV, treated vs. control, respectively, p<0.05). The reduction of the perfusion defect was associated with a significantly higher myocardial salvage index in the cell group as well as a significant increase in ejection fraction compared to control (EF at 8 weeks 43±7% vs. 35±3%, treated vs. control, respectively, p<0.05). This functional improvement was reflected by an increased wall thickness of the infarct and border zone in the treated group (11.2±2.2 mm) compared to control (8.6±1.6 mm, p<0.05) as well as an increased capillary density in the border zone (treated vs. control; 41.6±17.9 vs. 32.9±12.6 capillaries per 0.1 mm², p<0.05). CONCLUSIONS: This study demonstrates for the first time that recovery and intracoronary delivery of uncultured autologous tissue derived stromal cells at time of vessel reperfusion is feasible and improves ventricular function.

IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model.

Soft tissue loss presents an ongoing challenge in reconstructive surgery. Local stem cell application has recently been suggested as a possible novel therapy. In the present study we evaluated the potential of a silk fibroin-chitosan (SFCS) scaffold serving as a delivery vehicle for human adipose-derived stem cells (ASCs) in a murine soft tissue injury model. Green fluorescent protein (GFP)-labeled ASCs were seeded on SFCS scaffolds at a density of 1 x 10(5) ASCs per cm(2) for 48 hours and then suture-inlaid to a 6-mm, full-thickness skin defect in 6-week-old male athymic mice. Wound healing was tracked for 2 weeks by planimetry. Histology was evaluated at 2 and 4 weeks. Our data show that the extent of wound closure was significantly enhanced in the ASC-SFCS group versus SFCS and no-graft controls at postoperative day 8 (90% +/- 3% closure vs. 75% +/- 11% and 55% +/- 17%, respectively). Microvessel density at wound bed biopsy sites from 2 weeks postoperative was significantly higher in the ASC-SFCS group versus SFCS alone (7.5 +/- 1.1 vs. 5.1 +/- 1.0 vessels per high-power field). Engrafted stem cells were positive for the fibroblastic marker heat shock protein 47, smooth muscle actin, and von Willebrand factor at both 2 and 4 weeks. GFP-positive stem cells were also found to differentiate into epidermal epithelial cells at 4 weeks postoperative. In conclusion, human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound healing and show differentiation into fibrovascular, endothelial, and epithelial components of restored tissue.

Magnetic resonance imaging as a novel method of characterization of cutaneous photoaging in a murine model.

Environmental ultraviolet (UV) exposure exacts a significant toll annually in terms of overall morbidity and undesirable esthetic effects of skin aging. In order to establish the molecular and pathologic basis of this process, the murine model of ultraviolet B (UVB)-induced aging has long been used with reproducibility and success. Although morphometric and histological endpoints have been useful tools to describe this model historically, they fail to allow for the real time monitoring of the aging process, and do not fully account for effects of the in vivo environment on cutaneous strata with aging. The objective of the present study was to evaluate the ability of high-resolution magnetic resonance imaging (MRI) to characterize the murine model of photoaging throughout the aging process. Six-week-old male nu/nu mice were exposed to narrow-band UVB at 30 min intervals five times weekly for 10 weeks, resulting in a characteristic photoaged morphometric result. MRI scans were performed using a 7 tesla (7 T) small animal imaging platform at pre-exposure baseline at 4 and 10 weeks. Histological analysis of full-thickness biopsies taken in 10 week photoaged mice was correlated with MRI findings, and was compared against control animals receiving no ultraviolet radiation. MRI studies revealed a statistically significant prominent evolution of epidermal hyperplasia at 4 weeks versus baseline and at 10 weeks compared to 4 week values (0.172 +/- 0.017 mm versus undetectable, P < or = 0.05; 0.258 +/- 0.007 versus 0.172 +/- 0.017 mm, P < or = 0.05, respectively). A parallel trend of dermal hyperplasia which approached statistical significance was likewise noted at 10 weeks compared to baseline values (0.420 +/- 0.073 versus 0.295 +/- 0.078 mm, P = 0.06). Histology confirmed the progressive epidermal hyperplastic change characterized by MRI findings. This study validates the novel use of high-resolution MRI for study of the murine photoaging model and establishes its potential to describe progressive cutaneous pathology in such an experimental setting.

Dermal matrix as a carrier for in vivo delivery of human adipose-derived stem cells.

The aim of the present study was to evaluate the potential of acellular dermal matrix as a carrier for delivery of stem cells to the site of soft tissue defect in a murine skin injury model and to determine the potential of stem cells delivered via such an approach to successfully engraft, survive and differentiate locally. We showed that adipose-derived stem cells delivered via this matrix survived after in vivo engraftment, spontaneously differentiated along vascular endothelial, fibroblastic and epidermal epithelial lineages and significantly improved wound healing. Furthermore, an organ survey for transplanted cells showed no evidence of a systemic distribution beyond the cutaneous wound site, indicating that the adipose-derived stem cell-dermal matrix construct provides a novel and effective method for anatomically focused cellular therapy. In conclusion, stem cell-seeded dermal matrix is an effective means for targeted in vivo cell delivery for enhanced soft tissue regeneration.

VEGF is critical for spontaneous differentiation of stem cells into cardiomyocytes.

Cardiomyocyte regeneration is limited in adult life and is not sufficient to compensate for cell loss with myocardial infarction. Hence, the identification of a useful source of cardiomyocyte progenitors is of great interest for possible use in regenerative therapy. In this study, we isolated stem cells derived from human subcutaneous adipose tissue. The expression of Nkx2.5 and GATA-4 can be observed by PCR directly after extraction and during cultivation in some of these cells. Cardiac Troponin T and myosin light chain-2v become positive after 12 days of cultivation. To define respective factors responsible for spontaneous differentiation, we measured VEGF level in ADSC conditioned medium. Our data showed that ADSC secrete significant amount of VEGF (283.5pg per microgram DNA) and that anti-VEGF receptor antibodies blocked the cardiac differentiation. In conclusion, we demonstrated the spontaneous differentiation of human subcutaneous adipose-derived stem cells into a cardiomyocyte phenotype under standard culturing conditions.