Category Archives: Regenerative Medicine

Abstract

Mesenchymal stem cells (MSCs) encapsulation by three-dimensionally (3D) printed matrices were believed to provide a biomimetic microenvironment to drive differentiation into tissue-specific progeny, which made them a great therapeutic potential for regenerative medicine. Despite this potential, the underlying mechanisms of controlling cell fate in 3D microenvironments remained relatively unexplored. Here, we bioprinted a sweat gland (SG)like matrix to direct the conversion of MSC into functional SGs and facilitated SGs recovery in mice. By extracellular matrix differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator for SG specification. Our findings showed that Hmox1 could respond to the 3D structure activation and also be involved in MSC differentiation. Using inhibition and activation assay, CTHRC1 and Hmox1 synergistically boosted SG gene expression profile. Together, these findings indicated that biochemical and structural cues served as two critical impacts of 3D-printed matrix on MSC fate decision into the glandular lineage and functional SG recovery.

Mesenchymal stem cells (MSCs) hold great promise for therapeutic tissue engineering and regenerative medicine, largely because of their capacity for self-renewal and multipotent properties (1). However, their uncertain fate has a major impact on their envisioned therapeutic use. Cell fate regulation requires specific transcription programs in response to environmental cues (2, 3). Once stem cells are removed from their microenvironment, their response to environmental cues, phenotype, and functionality could often be altered (4, 5). In contrast to growing information concerning transcriptional regulation, guidance from the extracellular matrix (ECM) governing MSC identity and fate determination is not well understood. It remains an active area of investigation and may provide previously unidentified avenues for MSC-based therapy.

Over the past decade, engineering three-dimensional (3D) ECM to direct MSC differentiation has demonstrated great potential of MSCs in regenerative medicine (6). 3D ECM has been found to be useful in providing both biochemical and biophysical cues and to stabilize newly formed tissues (7). Culturing cells in 3D ECM radically alters the interfacial interactions with the ECM as compared with 2D ECM, where cells are flattened and may lose their differentiated phenotype (8). However, one limitation of 3D materials as compared to 2D approaches was the lack of spatial control over chemistry with 3D materials. One possible solution to this limitation is 3D bioprinting, which could be used to design the custom scaffolds and tissues (9).

In contrast to traditional engineering techniques, 3D cell printing technology is especially advantageous because it can integrate multiple biophysical and biochemical cues spatially for cellular regulation and ensure complex structures with precise control and high reproducibility. In particular, for our final goal of clinical practice, extrusion-based bioprinting may be more appropriate for translational application. In addition, as a widely used bioink for extrusion bioprinting, alginate-based hydrogel could maintain stemness of MSC due to the bioinert property and improve biological activity and printability by combining gelatin (10).

Sweat glands (SGs) play a vital role in thermal regulation, and absent or malfunctioning SGs in a hot environment can lead to hyperthermia, stroke, and even death in mammals (11, 12). Each SG is a single tube consisting of a functionally distinctive duct and secretory portions. It has low regenerative potential in response to deep dermal injury, which poses a challenge for restitution of lost cells after wound (13). A major obstacle in SG regeneration, similar to the regeneration of most other glandular tissues, is the paucity of viable cells capable of regenerating multiple tissue phenotypes (12). Several reports have described SG regeneration in vitro; however, dynamic morphogenesis was not identified nor was the overall function of the formed tissues explored (1416). Recent advances in bioprinting and tissue engineering led to the complexities in the matrix design and fabrication with appropriate biochemical cues and biophysical guidance for SG regeneration (1719).

Here, we adopted 3D bioprinting technique to mimic the regenerative microenvironment that directed the specific SG differentiation of MSCs and ultimately guided the formation and function of glandular tissue. We used alginate/gelatin hydrogel as bioinks in this present study due to its good cytocompatibility, printability, and structural maintenance in long-time culture. Although the profound effects of ECM on cell differentiation was well recognized, the importance of biochemical and structural cues of 3D-printed matrix that determined the cell fate of MSCs remained unknown; thus, the present study demonstrated the role of 3D-printed matrix cues on cellular behavior and tissue morphogenesis and might help in developing strategies for MSC-based tissue regeneration or directing stem cell lineage specification by 3D bioprinting.

The procedure for printing the 3D MSC-loaded construct incorporating a specific SG ECM (mouse plantar region dermis, PD) was shown schematically in Fig. 1A. A 3D cellular construct with cross section 30 mm 30 mm and height of 3 mm was fabricated by using the optimized process parameter (20). The 3D construct demonstrated a macroporous grid structure with hydrogel fibers evenly distributed according to the computer design. Both the width of the fibers and the gap between the fibers were homogeneous, and MSCs were embedded uniformly in the hydrogel matrix fibers to result in a specific 3D microenvironment. (Fig. 1B).

(A) Schematic description of the approach. (B) Full view of the cellular construct and representative microscopic and fluorescent images and the quantitative parameters of 3D-printed construct (scale bars, 200 m). Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Representative microscopy images of cell aggregates and tissue morphology at 3, 7, and 14 days of culture (scale bars, 50 m) and scanning electron microscopy (sem) images of 3D structure (scale bars, 20 m). PD+/PD, 3D construct with and without PD. (D) DNA contents, collagen, and GAGs of native tissue and PD. (E) Proliferating cells were detected through Ki67 stain at 3, 7, and 14 days of culture. (F) Live/dead assay show cell viability at days 3, 7, and 14. *P < 0.05.

During the maintenance of constructs for stem cell expansion, MSCs proliferated to form aggregates of cells but self-assembled to an SG-like structure only with PD administration (Fig. 1C and fig. S1, A to C). We carried out DNA quantification assay to evaluate the cellular content in PD and found the cellular matrix with up to 90% reduction, only 3.4 0.7 ng of DNA per milligram tissue remaining in the ECM. We also estimated the proportions of collagen and glycosaminoglycans (GAGs) in ECM through hydroxyproline assay and dimethylmethylene blue assay, the collagen contents could increase to 112.6 11.3%, and GAGs were well retained to 81 9.6% (Fig. 1D). Encapsulated cells were viable, with negligible cell death apparent during extrusion and ink gelation by ionic cross-linking, persisting through extended culture in excess of 14 days. The fluorescence intensity of Ki67 of MSCs cultured in 2D condition decreased from days 3 (152.7 13.4) to 14 (29.4 12.9), while maintaining higher intensity of MSCs in 3D construct (such as 211.8 19.4 of PD+3D group and 209.1 22.1 of PD3D group at day 14). And the cell viability in 3D construct was found to be sufficiently high (>80%) when examined on days 3, 7, and 14. The phenomenon of cell aggregate formation and increased cell proliferation implied the excellent cell compatibility of the hydrogel-based construct and promotion of tissue development of 3D architectural guides, which did not depend on the presence or absence of PD (Fig. 1, E and F).

The capability of 3D-printed construct with PD directing MSC to SGs in vitro was investigated. The 3D construct was dissolved, and cells were isolated at days 3, 7, and 14 for transcriptional analysis. Expression of the SG markers K8 and K18 was higher from the 3D construct with (3D/PD+) than without PD (3D/PD); K8 and K18 expression in the 3D/PD construct was similar to with control that MSCs cultured in 2D condition, which implied the key role of PD in SG specification. As compared with the 2D culture condition, 3D administration (PD+) up-regulated SG markers, which indicated that the 3D structure synergistically boosted the MSC differentiation (Fig. 2A).

(A) Transcriptional expression of K8, K18, Fxyd2, Aqp5, and ATP1a1 in 3D-bioprinted cells with and without PD in days 3, 7, and 14 culture by quantitative real-time polymerase chain reaction (qRT-PCR). Data are means SEM. (B) Comparison of SG-specific markers K8 and K18 in 3D-bioprinted cells with and without PD (K8 and K18, red; DAPI, blue; scale bars, 50 m). (C and D) Comparison of SG secretion-related markers ATP1a1 (C) and Ca2+ (D) in 3D-bioprinted cells with and without PD [ATP1a1 and Ca2+, red; 4,6-diamidino-2-phenylindole (DAPI), blue; scale bars, 50 m].

In addition, we tested secretion-related genes to evaluate the function of induced SG cells (iSGCs). Although levels of the ion channel factors of Fxyd2 and ATP1a1 were increased notably in 2D culture with PD and ATP1a1 up-regulated in the 3D/PD construct, all the secretory genes of Fxyd2, ATP1a1, and water transporter Aqp5 showed the highest expression level in the 3D/PD+ construct (Fig. 2A). Considering the remarkable impact, further analysis focused on 3D constructs.

Immunofluorescence staining confirmed the progression of MSC differentiation. At day 7, cells in the 3D/PD+ construct began to express K8 and K18, which was increased at day 14, whereas cells in the 3D/PD construct did not express K8 and K18 all the time (Fig. 2B and fig. S2A). However, the expression of ATP1a1 (ATPase Na+/K+ transporting subunit alpha 1) and free Ca2+ concentration did not differ between cells in the 3D/PD+ and 3D/PD constructs (Fig. 2, C and D). By placing MSCs in such a 3D environment, secretion might be stimulated by rapid cell aggregation without the need for SG lineage differentiation. Cell aggregationimproved secretion might be due to the benefit of cell-cell contact (fig. S2B) (21, 22).

To map the cell fate changes during the differentiation between MSCs and SG cells, we monitored the mRNA levels of epithelial markers such as E-cadherin, occludin, Id2, and Mgat3 and mesenchymal markers N-cadherin, vimentin, Twist1, and Zeb2. The cells transitioned from a mesenchymal status to a typical epithelial-like status accompanied by mesenchymal-epithelial transition (MET), then epithelial-mesenchymal transition (EMT) occurred during the further differentiation of epithelial lineages to SG cells (fig. S3A). In addition, MET-related genes were dynamically regulated during the SG differentiation of MSCs. For example, the mesenchymal markers N-cadherin and vimentin were down-regulated from days 1 to 7, which suggested cells losing their mesenchymal phenotype, then were gradually up-regulated from days 7 to 10 in their response to the SG phenotype and decreased at day 14. The epithelial markers E-cadherin and occludin showed an opposite expression pattern: up-regulated from days 1 to 5, then down-regulated from days 7 to 10 and up-regulated again at day 14. The mesenchymal transcriptional factors ZEB2 and Twist1 and epithelial transcriptional factors Id2 and Mgat3 were also dynamically regulated.

We further analyzed the expression of these genes at the protein level by immunofluorescence staining (figs. S3B and S4). N-cadherin was down-regulated from days 3 to 7 and reestablished at day 14, whereas E-cadherin level was increased from days 3 to 7 and down-regulated at day 14. Together, these results indicated that a sequential and dynamic MET-EMT process underlie the differentiation of MSCs to an SG phenotype, perhaps driving differentiation more efficiently (23). However, the occurrence of the MET-EMT process did not depend on the presence of PD. Thus, a 3D structural factor might also participate in the MSC-specific differentiation (fig. S3C).

To investigate the underlying mechanism of biochemical cues in lineage-specific cell fate, we used quantitative proteomics analysis to screen the ECM factors differentially expressed between PD and dorsal region dermis (DD) because mice had eccrine SGs exclusively present in the pads of their paws, and the trunk skin lacks SGs. In total, quantitative proteomics analyses showed higher expression levels of 291 proteins in PD than DD. Overall, 66 were ECM factors: 23 were significantly up-regulated (>2-fold change in expression). We initially determined the level of proteins with the most significant difference after removing keratins and fibrin: collagen triple helix repeat containing 1 (CTHRC1) and thrombospondin 1 (TSP1) (fig. S5). Western blotting was performed to further confirm the expression level of CTHRC1 and TSP1, and we then confirmed that immunofluorescence staining at different developmental stages in mice revealed increased expression of CTHRC1 in PD with SG development but only slight expression in DD at postnatal day 28, while TSP1 was continuously expressed in DD and PD during development (Fig. 3, A to C). Therefore, TSP1 was required for the lineage-specific function during the differentiation in mice but was not dispensable for SG development.

(A and B) Differential expression of CTHRC1 and TSP1in PD and back dermis (DD) ECM of mice by proteomics analysis (A) and Western blotting (B). (C) CTHRC1 and TSP1 expression in back and plantar skin of mice at different developmental times. (Cthrc1/TSP1, red; DAPI, blue; scale bars, 50 m).

According to previous results of the changes of SG markers, 3D structure and PD were both critical to SG fate. Then, we focused on elucidating the mechanisms that underlie the significant differences observed in 2D and 3D conditions with or without PD treatment. To this end, we performed transcriptomics analysis of MSCs, MSCs treated with PD, MSCs cultured in 3D construct, and MSC cultured in 3D construct with PD after 3-day treatment. We noted that the expression profiles of MSCs treated with 3D, PD, or 3D/PD were distinct from the profiles of MSCs (Fig. 4A). Through Gene Ontology (GO) enrichment analysis of differentially expressed genes, it was shown that PD treatment in 2D condition induced up-regulation of ECM and inflammatory response term, and the top GO term for MSCs in 3D construct was ECM organization and extracellular structure organization. However, for the MSCs with 3D/PD treatment, we found very significant overrepresentation of GO term related to branching morphogenesis of an epithelial tube and morphogenesis of a branching structure, which suggested that 3D structure cues and biochemical cues synergistically initiate the branching of gland lineage (fig S6). Heat maps of differentially expressed ECM organization, cell division, gland morphogenesis, and branch morphogenesis-associated genes were shown in fig. S7. To find the specific genes response to 3D structure cues facilitating MSC reprogramming, we analyzed the differentially expressed genes of four groups of cells (Fig. 4B). The expression of Vwa1, Vsig1, and Hmox1 were only up-regulated with 3D structure stimulation, especially the expression of Hmox1 showed a most significant increase and even showed a higher expression addition with PD, which implied that Hmox1 might be the transcriptional driver of MSC differentiation response to 3D structure cues. Differential expression of several genes was confirmed by quantitative polymerase chain reaction (qPCR): Mmp9, Ptges, and Il10 were up-regulated in all the treated groups. Likewise, genes involving gland morphogenesis and branch morphogenesis such as Bmp2, Tgm2, and Sox9 showed higher expression in 3D/PD-treated group. Bmp2 was up-regulated only in 3D/PD-treated group, combined with the results of GO analysis, we assumed that Bmp2 initiated SG fate through inducing branch morphogenesis and gland differentiation (Fig. 4C).

(A) Gene expression file of four groups of cells (R2DC, MSCs; R2DT, MSC with PD treatment; R3DC, MSC cultured in 3D construct; and R3DT, MSC treated with 3D/PD). (B) Up-regulated genes after treatment (2DC, MSCs; 2DT, MSC with PD treatment; 3DC, MSC cultured in 3D construct; and 3DT, MSC treated with 3D/PD). (C) Differentially expressed genes were further validated by RT-PCR analysis. [For all RT-PCR analyses, gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with 40 cycles, data are represented as the means SEM, and n = 3].

To validate the role of HMOX1 and CTHRC1 in the differentiation of MSCs to SG lineages, we analyzed the gene expression of Bmp2 by regulating the expression of Hmox1 and CTHRC1 based on the 3D/PD-treated MSCs. The effects of caffeic acid phenethyl ester (CAPE) and tin protoporphyrin IX dichloride (Snpp) on the expression of Hmox1 were evaluated by quantitative real-time (qRT)PCR. Hmox1 expression was significantly activated by CAPE and reduced by Snpp. Concentration of CTHRC1 was increased with recombinant CTHRC1 and decreased with CTHRC1 antibody. That is, it was negligible of the effects of activator and inhibitor of Hmox1 and CTHRC1 on cell proliferation (fig. S8, A and B). Hmox1 inhibition or CTHRC1 neutralization could significantly reduce the expression of Bmp2, while Hmox1 activation or increased CTHRC1 both activated Bmp2 expression. Furthermore, Bmp2 showed highest expression by up-regulation of Hmox1 and CTHRC1 simultaneously and sharply decreased with down-regulation of Hmox1 and CTHRC1 at the same time (Fig. 5A). Immunofluorescent staining revealed that the expression of bone morphogenetic protein 2 (BMP2) at the translational level with CTHRC1 and Hmox1 regulation showed a similar trend with transcriptional changes (Fig. 5B). Likewise, the expression of K8 and K18 at transcriptional and translational level changed similarly with CTHRC1 and Hmox1 regulation (fig. S9, A and B). These results suggested that CTHRC1 and Hmox1 played an essential role in SG fate separately, and they synergistically induced SG direction from MSCs (Fig. 5C).

(A and B) Transcriptional analysis (A) and translational analysis (PD, MSCs; PD+, MSCs with 3D/PD treatment; CAPE, MSCs treated with 3D/PD and Hmox1 activator; Snpp, MSCs treated with 3D/PD and Hmox1 inhibitor; Cthrc1, MSCs treated with 3D/PD and recombinant CTHRC1; anti, MSCs treated with 3D/PD and CTHRC1 antibody: +/+, MSCs treated with 3D/PD and Hmox1 activator and recombinant CTHRC1; and /, MSCs treated with 3D/PD and Hmox1 inhibitor and CTHRC1 antibody. Data are represented as the means SEM and n = 3) (B) of bmp2 with regulation of CTHRC1 and Hmox1. (C) The graphic illustration of 3D-bioprinted matrix directed MSC differentiation. CTHRC1 is the main biochemical cues during SG development, and structural cues up-regulated the expression of Hmox1 synergistically initiated branching morphogenesis of SG. *P < 0.05.

Next, we sought to assess the repair capacity of iSGCs for in vivo implications, the 3D-printed construct with green fluorescent protein (GFP)labeled MSCs was transplanted in burned paws of mice (Fig. 6A). We measured the SG repair effects by iodine/starch-based sweat test at day 14. Only mice with 3D/PD treatment showed black dots on foot pads (representing sweating), and the number increased within 10 min; however, no black dots were observed on untreated and single MSC-transplanted mouse foot pads even after 15 min (Fig. 6B). Likewise, hematoxylin and eosin staining analysis revealed SG regeneration in 3D/PD-treated mice (Fig. 6C). GFP-positive cells were characterized as secretory lumen expressing K8, K18, and K19. Of note, the GFP-positive cells were highly distributed in K14-positive myoepithelial cells of SGs but were absent in K14-positive repaired epidermal wounds (Fig. 6, D and E). Thus, differentiated MSCs enabled directed restitution of damaged SG tissues both at the morphological and functional level.

(A) Schematic illustration of approaches for engineering iSGCs and transplantation. (B) Sweat test of mice treated with different cells. Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Histology of plantar region without treatment and transplantation of MSCs and iSGCs (scale bars, 200 m). (D) Involvement of GFP-labeled iSGCs in directed regeneration of SG tissue in thermal-injured mouse model (K14, red; GFP, green; DAPI, blue; scale bar, 200 m). (E) SG-specific markers K14, K19, K8, and K18 detected in regenerated SG tissue (arrows). (K14, K19, K8, and K18, red; GFP, green; scale bars, 50 m).

A potential gap in MSC-based therapy still exists between current understandings of MSC performance in vivo in their microenvironment and their intractability outside of that microenvironment (24). To regulate MSCs differentiation into the right phenotype, an appropriate microenvironment should be created in a precisely controlled spatial and temporal manner (25). Recent advances in innovative technologies such as bioprinting have enabled the complexities in the matrix design and fabrication of regenerative microenvironments (26). Our findings demonstrated that directed differentiation of MSCs into SGs in a 3D-printed matrix both in vitro and in vivo was feasible. In contrast to conventional tissue-engineering strategies of SG regeneration, the present 3D-printing approach for SG regeneration with overall morphology and function offered a rapid and accurate approach that may represent a ready-to-use therapeutic tool.

Furthermore, bioprinting MSCs successfully repaired the damaged SG in vivo, suggesting that it can improve the regenerative potential of exogenous differentiated MSCs, thereby leading to translational applications. Notably, the GFP-labeled MSC-derived glandular cells were highly distributed in K14-positive myoepithelial cells of newly formed SGs but were absent in K14-positive repaired epidermal wounds. Compared with no black dots were observed on single MSC-transplanted mouse foot pads, the black dots (representing sweating function) can be observed throughout the entire examination period, and the number increased within 10 min on MSC-bioprinted mouse foot pads. Thus, differentiated MSCs by 3D bioprinting enabled exclusive restitution of damaged SG tissues morphologically and functionally.

Although several studies indicated that engineering 3D microenvironments enabled better control of stem cell fates and effective regeneration of functional tissues (2730), there were no studies concerning the establishment of 3D-bioprinted microenvironments that can preferentially induce MSCs differentiating into glandular cells with multiple tissue phenotypes and overall functional tissue. To find an optimal microenvironment for promoting MSC differentiation into specialized progeny, biochemical properties are considered as the first parameter to ensure SG specification. In this study, we used mouse PD as the main composition of a tissue-specific ECM. As expected, this 3D-printed PD+ microenvironment drove the MSC fate decision to enhance the SG phenotypic profile of the differentiated cells. By ECM differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator of 3D-printed matrix for SG specification. TSP1 was required for the lineage-specific function during the differentiation in mice but was not dispensable for SG development. Thus, we identified CTHRC1 as a specific factor during SG development. To our knowledge, this is the first demonstration of CTHRC1 involvement in dictating MSC differentiation to SG, highlighting a potential therapeutic tool for SG injury.

The 3D-printed matrix also provided architectural guides for further SG morphogenesis. Our results clearly show that the 3D spatial dimensionality allows for better cell proliferation and aggregation and affect the characteristics of phenotypic marker expression. Notably, the importance of 3D structural cues on MSC differentiation was further proved by MET-EMT process during differentiation, where the influences did not depend on the presence of biochemical cues. To fully elucidate the underlying mechanisms, we first examined how 3D structure regulating stem cell fate choices. According to our data, Hmox1 is highly up-regulated in 3D construct, which were supposed to response to hypoxia, with a previously documented role in MSC differentiation (31, 32). It is suggested that 3D microenvironment induced rapid cell aggregation leading to hypoxia and then activated the expression of Hmox1.

Through regulation of the expression of Hmox1 and addition or of CTHRC1 in the matrix, we confirmed that each of them is critical for SG reprogramming, respectively. Thus, biochemical and structural cues of 3D-printed matrix synergistically creating a microenvironment could enhance the accuracy and efficiency of MSC differentiation, thereby leading to resulting SG formation. Although we further need a more extensive study examining the role of other multiple cues and their possible overlap function in regulating MSC differentiation, our findings suggest that CTHRC1 and Hmox1 provide important signals that cooperatively modulate MSC lineage specification toward sweat glandular lineage. The 3D structure combined with PD stimulated the GO functional item of branch morphogenesis and gland formation, which might be induce by up-regulation of Bmp2 based on the verification of qPCR results. Although our results could not rule out the involvement of other factors and their possible overlapping role in regulating MSC lineage specification toward SGs, our findings together with several literatures suggested that BMP2 plays a critical role in inducing branch morphogenesis and gland formation (3335).

In summary, our findings represented a novel strategy of directing MSC differentiation for functional SG regeneration by using 3D bioprinting and pave the way for a potential therapeutic tool for other complex glandular tissues as well as further investigation into directed differentiation in 3D conditions. Specifically, we showed that biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation, and our results highlighted the importance of 3D-printed matrix cues as regulators of MSC fate decisions. This avenue opens up the intriguing possibility of shifting from genetic to microenvironmental manipulations of cell fate, which would be of particular interest for clinical applications of MSC-based therapies.

The main aim and design of the study was first to determine whether by using 3D-printed microenvironments, MSCs can be directed to differentiate and regenerate SGs both morphologically and functionally. Then, to investigate the underlying molecular mechanism of biochemical and structural cues of 3D-printed matrix involved in MSCs reprogramming. The primary aims of the study design were as follows: (i) cell aggregation and proliferation in a 3D-bioprinted construct; (ii) differentiation of MSCs at the cellular phenotype and functional levels in the 3D-bioprinted construct; (iii) the MET-EMT process during differentiation; (iv) differential protein expression of the SG niche in mice; (v) differential genes expression of MSCs in 3D-bioprinted construct; (vi) the key role of CTHRC1 and HMOX1 in MSCs reprogramming to SGCs; and (vii) functional properties of regenerated SG in vivo.

Gelatin (Sigma-Aldrich, USA) and sodium alginate (Sigma-Aldrich, USA) were dissolved in phosphate-buffered saline (PBS) at 15 and 1% (w/v), respectively. Both solutions were sterilized under 70C for 30 min three times at an interval of 30 min. The sterilized solutions were packed into 50-ml centrifuge tubes, stored at 4C, and incubated at 37C before use.

From wild-type C57/B16 mice (Huafukang Co., Beijing) aged 5 days old, dermal homogenates were prepared by homogenizing freshly collected hairless mouse PD with isotonic phosphate buffer (pH 7.4) for 20 min in an ice bath to obtain 25% (w/v) tissue suspension. The supernatant was obtained after centrifugation at 4C for 20 min at 10,000g. The DNA content was determined using Hoechst 33258 assay (Beyotime, Beijing). The fluorescence intensity was measured to assess the amount of remaining DNA within the decellularized ECMs and the native tissue using a fluorescence spectrophotometer (Thermo Scientific, Evolution 260 Bio, USA). The GAGs content was estimated via 1,9-dimethylmethylene blue solution staining. The absorbance was measured with microplate reader at wavelength of 492 nm. The standard curve was made using chondroitin sulfate A. The total COL (Collagen) content was determined via hydroxyproline assay. The absorbance of the samples was measured at 550 nm and quantified by referring to a standard curve made with hydroxyproline.

MSCs were bioprinted with matrix materials by using an extrusion-based 3D bioprinter (Regenovo Co., Bio-Architect PRO, Hangzhou). Briefly, 10 ml of gelatin solution (10% w/v) and 5 ml of alginate solution (2% w/v) were warmed under 37C for 20 min, gently mixed as bioink and used within 30 min. MSCs were collected from 100-mm dishes, dispersed into single cells, and 200 l of cell suspension was gently mixed with matrix material under room temperature with cell density 1 million ml1. PD (58 g/ml) was then gently mixed with bioink. Petri dishes at 60 mm were used as collecting plates in the 3D bioprinting process. Within a temperature-controlled chamber of the bioprinter, with temperature set within the gelation region of gelatin, the mixture of MSCs and matrix materials was bioprinted into a cylindrical construct layer by layer. The nozzle-insulation temperature and printing chamber temperature were set at 18 and 10C, respectively; nozzles with an inner diameter of 260 m were chosen for printing. The diameter of the cylindrical construct was 30 mm, with six layers in height. After the temperature-controlled bioprinting process, the printed 3D constructs were immersed in 100-mM calcium chloride (Sigma-Aldrich, USA) for 3 min for cross-linking, then washed with Dulbeccos modified Eagle medium (DMEM) (Gibco, USA) medium for three times. The whole printing process was finished in 10 min. The 3D cross-linked construct was cultured in DMEM in an atmosphere of 5% CO2 at 37C. The culture medium was changed to SG medium [contains 50% DMEM (Gibco, New York, NY) and 50% F12 (Gibco) supplemented with 5% fetal calf serum (Gibco), 1 ml/100 ml penicillin-streptomycin solution, 2 ng/ml liothyronine sodium (Gibco), 0.4 g/ml hydrocortisone succinate (Gibco), 10 ng/ml epidermal growth factor (PeproTech, Rocky Hill, NJ), and 1 ml/100 ml insulin-transferrin-selenium (Gibco)] 2 days later. The cell morphology was examined and recorded under an optical microscope (Olympus, CX40, Japan).

Fluorescent live/dead staining was used to determine cell viability in the 3D cell-loaded constructs according to the manufacturers instructions (Sigma-Aldrich, USA). Briefly, samples were gently washed in PBS three times. An amount of 1 M calcein acetoxymethyl (calcein AM) ester (Sigma-Aldrich, USA) and 2 M propidium iodide (Sigma-Aldrich, USA) was used to stain live cells (green) and dead cells (red) for 15 min while avoiding light. A laser scanning confocal microscopy system (Leica, TCSSP8, Germany) was used for image acquisition.

The cell-printed structure was harvested and fixed with a solution of 4% paraformaldehyde. The structure was embedded in optimal cutting temperature (OCT) compound (Sigma-Aldrich, USA) and sectioned 10-mm thick by using a cryotome (Leica, CM1950, Germany). The sliced samples were washed repeatedly with PBS solution to remove OCT compound and then permeabilized with a solution of 0.1% Triton X-100 (Sigma-Aldrich, USA) in PBS for 5 min. To reduce nonspecific background, sections were treated with 0.2% bovine serum albumin (Sigma-Aldrich, USA) solution in PBS for 20 min. To visualize iSGCs, sections were incubated with primary antibody overnight at 4C for anti-K8 (1:300), anti-K14 (1:300), anti-K18 (1:300), anti-K19 (1:300), anti-ATP1a1 (1:300), anti-Ki67 (1:300), antiN-cadherin (1:300), antiE-cadherin (1:300), anti-CTHRC1 (1:300), or anti-TSP1 (1:300; all Abcam, UK) and then incubated with secondary antibody for 2 hours at room temperature: Alexa Fluor 594 goat anti-rabbit (1:300), fluorescein isothiocyanate (FITC) goat anti-rabbit (1:300), FITC goat anti-mouse (1:300), or Alexa Fluor 594 goat anti-mouse (1:300; all Invitrogen, CA). Sections were also stained with 4,6-diamidino-2-phenylindole (Beyotime, Beijing) for 15 min. Stained samples were visualized, and images were captured under a confocal microscope.

To harvest the cells in the construct, the 3D constructs were dissolved by adding 55 mM sodium citrate and 20 mM EDTA (Sigma-Aldrich, USA) in 150 mM sodium chloride (Sigma-Aldrich, USA) for 5 min while gently shaking the petri dish for better dissolving. After transfer to 15-ml centrifuge tubes, the cell suspensions were centrifuged at 200 rpm for 3 min, and the supernatant liquid was removed to harvest cells for further analysis.

Total RNA was isolated from cells by using TRIzol reagent (Invitrogen, USA) following the manufacturers protocol. RNA concentration was measured by using a NanoPhotometer (Implen GmbH, P-330-31, Germany). Reverse transcription involved use of a complementary DNA synthesis kit (Takara, China). Gene expression was analyzed quantitatively by using SYBR green with the 7500 Real-Time PCR System (Takara, China). The primers and probes for genes were designed on the basis of published gene sequences (table S1) (National Center for Biotechnology Information and PubMed). The expression of each gene was normalized to that for glyceraldehyde-3-phosphate dehydrogenase and analyzed by the 2-CT method. Each sample was assessed in triplicate.

The culture medium was changed to SG medium with 2 mM CaCl2 for at least 24 hours, and cells were loaded with fluo-3/AM (Invitrogen, CA) at a final concentration of 5 M for 30 min at room temperature. After three washes with calcium-free PBS, 10 M acetylcholine (Sigma-Aldrich, USA) was added to cells. The change in the Fluo 3 fluorescent signal was recorded under a laser scanning confocal microscopy.

Cell proliferation was evaluated through CCK-8 (Cell counting kit-8) assay. Briefly, cells were seeded in 96-well plates at the appropriate concentration and cultured at 37C in an incubator for 4 hours. When cells were adhered, 10 l of CCK-8 working buffer was added into the 96-well plates and incubated at 37C for 1 hour. Absorbance at 450 nm was measured with a microplate reader (Tecan, SPARK 10M, Austria).

Proteomics of mouse PD and DD involved use of isobaric tags for relative and absolute quantification (iTRAQ) in BGI Company, with differentially expressed proteins detected in PD versus DD. Twofold greater difference in expression was considered significant for further study.

Tissues were grinded and lysed in radioimmunoprecipitation assay buffer (Beyotime, Nanjing). Proteins were separated by 12% SDSpolyacrylamide gel electrophoresis and transferred to a methanol-activated polyvinylidene difluoride membrane (GE Healthcare, USA). The membrane was blocked for 1 hour in PBS with Tween 20 containing 5% bovine serum albumin (Sigma-Aldrich, USA) and probed with the antibodies anti-CTHRC1 (1:1000) and anti-TSP1 (1:1000; both Abcam, UK) overnight at 4C. After 2 hours of incubation with goat anti-rabbit horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology, CA), the protein bands were detected by using luminal reagent (GE Healthcare, ImageQuant LAS 4000, USA).

Total RNA was prepared with TRIzol (Invitrogen), and RNA sequencing was performed using HiSeq 2500 (Illumina). Genes with false discovery rate < 0.05, fold difference > 2.0, and mean log intensity > 2.0 were considered to be significant.

CAPE or Snpp was gently mixed with bioink at a concentration of 10 M. Physiological concentration of CTHRC1 was measured by enzyme linked immunosorbent assay (ELISA) (80 ng/ml), and then recombinant CTHRC1 or CTHRC1 antibody was added into the bioink at a concentration of 0.4 g/ml. The effect of inhibitor and activator was estimated by qRT-PCR or ELISA.

Mice were anesthetized with pentobarbital (100 mg/kg) and received subcutaneous buprenorphine (0.1 mg/kg) preoperatively. Full-thickness scald injuries were created on paw pads with soldering station (Weller, WSD81, Germany). Mice recovered in clean cages with paper bedding to prevent irritation or infection. Mice were monitored daily and euthanized at 30 days after wounding. Mice were maintained in an Association for Assessment and Accreditation of Laboratory Animal Careaccredited animal facility, and procedures were performed with Institutional Animal Care and Use Committeeapproved protocols.

MSCs in 3D-printed constructs with PD were cultured with DMEM for 2 days and then replaced with SG medium. The SG medium was changed every 2 days, and cells were harvested on day 12. The K18+ iSGCs were sorting through flow cytometry and injected into the paw pads (1 106 cells/50 l) of the mouse burn model by using Microliter syringes (Hamilton, 7655-01, USA). Then, mice were euthanized after 14 days; feet were excised and fixed with 10% formalin (Sigma-Aldrich, USA) overnight for paraffin sections and immunohistological analysis.

The foot pads of anesthetized treated mice were first painted with 2% (w/v) iodine/ethanol solution then with starch/castor oil solution (1 g/ml) (Sigma-Aldrich, USA). After drying, 50 l of 100 M acetylcholine (Sigma-Aldrich, USA) was injected subcutaneously into paws of mice. Pictures of the mouse foot pads were taken after 5, 10, and 15 min.

All data were presented as means SEM. Statistical analyses were performed using GraphPad Prism7 statistical software (GraphPad, USA). Significant differences were calculated by analysis of variance (ANOVA), followed by the Bonferroni test when performing multiple comparisons between groups. P < 0.05 was considered as a statistically significant difference.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz1094/DC1

Fig. S1. Biocompatibility of 3D-bioprinted construct and cellular morphology in 2D monolayer culture.

Fig. S2. Expression of SG-specific and secretion-related markers in MSCs and SG cells in vitro.

Fig. S3. Transcriptional and translational expression of epithelial and mesenchymal markers in 3D-bioprinted cells with and without PD.

Fig. S4. Expression of N- and E-cadherin in MSCs and SG cells in 2D monolayer culture.

Fig. S5. Proteomic microarray assay of differential gene expression between PD and DD ECM in postnatal mice.

Fig. S6. GO term analysis of differentially expressed pathways.

Fig. S7. Heat maps illustrating differential expression of genes implicated in ECM organization, cell division, and gland and branch morphogenesis.

Fig. S8. The expression of Hmox1 and the concentration of CTHRC1 on treatment and the related effects on cell proliferation.

Fig. S9. The expression of K8 and K18 with Hmox1 and CTHRC1 regulation.

Table S1. Primers for qRT-PCR of all the genes.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: Funding: This study was supported in part by the National Nature Science Foundation of China (81571909, 81701906, 81830064, and 81721092), the National Key Research Development Plan (2017YFC1103300), Military Logistics Research Key Project (AWS17J005), and Fostering Funds of Chinese PLA General Hospital for National Distinguished Young Scholar Science Fund (2017-JQPY-002). Author contributions: B.Y. and S.H. were responsible for the design and primary technical process, conducted the experiments, collected and analyzed data, and wrote the manuscript. Y.W. and R.W. helped perform the main experiments. Y.Z. and T.H. participated in the 3D printing. W.S. and Z.L. participated in cell experiments and postexamination. S.H. and X.F. collectively oversaw the collection of data and data interpretation and revised the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration - Science...

In what initially sounds like a blend of Frankenstein and Honey, I Shrunk the Kids, scientists at the Wake Forest Institute for Regenerative Medicine (WFIRM) have built the worlds most advanced model of a human body ever created in a lab. Complete with a system of organs, such as heart, liver and lungs, that function a whole lot like the real thing, the body model was built to replicate as closely as possible the reactions of an actual human body. The difference? Each miniaturized organ is around one-millionth the size of a real-life adult organ.

To create the model, tiny samples of human tissue cells are isolated and engineered into miniature versions of the human organ, Anthony Atala, director of WFIRM, told Digital Trends. They can contain blood vessel cells, immune system cells, and even fibroblasts, the cells in the connective tissue. We designed the integrated platform, or multi-tissue chip, supporting multiple tissue types under a common recirculating fluid. The system, depending on how many tissues it uses, can be designed to fit an area about the size of a deck of cards.

The body doesnt actually look like a body. Instead, its a series of chips and microfluidic devices. The purpose wasnt to build a recognizable miniature human, but rather a testing platform which functions and reacts like the body its based on. For instance, each organ contains blood vessel cells, immune system cells, and fibroblasts, the cells found in connective tissue. The heart beats around 60 times per minute, the lung breathes air from its surroundings, and the liver is used to break down toxic compounds into waste products.

The aim of building this test platform is to better predict and test how a human will react to new drug treatments. This can be used to detect harmful and adverse effects of drugs during the development stage before they enter clinical trials in patients. This can help speed up the process of bringing drugs to market, while also lowering the cost of clinical trials and reducing or even eliminating animal testing.

As part of their research, the team tested the system by using it to screen 10 drugs taken off the market by the U.S. Food and Drug Administration. When these drugs were tested in cell culture, animals and human clinical trials, no negative side effects were identified until they were being widely used by humans. At that point, it was discovered that the drugs could be harmful to people. The system developed at WFIRM was able to detect this toxicity, matching the damage found in humans.

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Researchers build the worlds most sophisticated lab model of the human body - Digital Trends

Tissue engineering combines the field of cell biology with material science in order to generate tissues and organs that may be used for regeneration, replacement or reconstruction of human bodies. In the past 10 years, there has been an exponential growth in these therapies, with great optimism and excitement about the potential effects or implications.

Since the end of the 20th century, cultured urethral mucosa cells have been used for repair of hypospadias, a congenital malformation of the urinary tract. In a survey published in 2019, tissue-engineered grafts showed even better results when used in children for primary hypospadias repair than in adults for urethral stricture repair.

Recently, a breakthrough in the surgical treatment of male urethral stricture was reported when a total of 65 patients with urethral strictures successfully were treated with MukoCell, a tissue-engineered oral mucosa transplant. With a mean follow-up of 12.1 months, recurrence was observed in only 12 patients. This corresponds to a success rate of 81.5%.About 1% of the male population suffers from strictures of the urethra.

Patients are chronically ill, with severely diminished quality of life, suffering from low urinary flow, pain, chronic urinary infections, urinary stones, urinary reflux, and damage to and failure of the urinary system. If left untreated, life-threatening urinary retention can occur.

The gold standard for urethral reconstruction is represented by the use of oral mucosa graft, with success rates reported in literature of around 80%. However, due to the complication rate at the mucosa harvest site, only a minority of operative urologists carry out this procedure.

It requires the excision of large segments of mucosa from the mouth of the patients. This severe damage to healthy tissue frequently is accompanied by multiple injuries with a significant impact on patients quality of life intraoral pain, bleeding, swelling, sensory loss and oral numbness which in many cases are persistent.

Other long term consequences include compromised oral health, scarring, chronic ulcers due to repeated bites on scar bulges, impaired lip mobility, permanent salivation, oral stenosis, facial deformities, diminished facial expressions, impaired mouth opening and impaired drinking, eating and speaking, periodontal disease; and loss of teeth and implants. One of the late consequences resulting from chronic irritation and inflammation is the increased risk of oral cancer.

Because of these risks and complications, many doctors and patients refuse this operation. Moreover, in certain situations this operation cannot be performed, such as where the patient only has a small oral cavity or limited mouth opening capacity, meaning access to the oral cavity is limited and excision of larger pieces of oral mucosa is not possible.

A significant proportion of patients are not willing to undergo the excision of oral grafts, including patients with tendency to increased scar formation, where the excision of oral mucosa is associated with risks of parafunctional bites, chronic irritation and inflammation; or patients with dentures, where the excision may lead to poorly fitting dentures or loss of dental implants. This counts even more if there is pre-existing oral mucosal damage, for example after previous removal of oral mucosa.

For other patients, the oral complications cannot be tolerated because impairment of physiognomy, oral anatomy or gustatory sensation impacts their job or social function; such as teachers, singers, politicians, actors, speakers, salespeople, cooks and musicians who play wind or brass instruments.

Tissue-engineered transplants represent the group of advanced tissue-engineered therapies (ATMPs). These are subject to EU regulation; in order to obtain market access, they must receive authorisation from the European Medicines Agency (EMA).

In order to obtain this approval, high standards must be met regarding proof of the quality, safety and efficacy of these products. Although tissue-engineered products may have a high impact on patients health, only a few of them will be approved. Tissue engineering techniques are complex and require a high standard of specialised laboratories.

Regarding quality and safety, MukoCell has already received a certificate from the EMA. MukoCell is manufactured in a state of the art cell culture factory, which has been specifically designed for engineering of tissue especially for medical use and complies with GMP guidelines for the production of pharmaceuticals. The manufacturing process starts with a tiny biopsy from the oral mucosa of the patient.

Oral mucosa is easily accessible in any patient; and biopsy under simple local anaesthesia is easy, non-invasive and painless for patients. The tissue is sent to the tissue factory where the biopsy is explanted in cell culture media. Cells are grown out and undergo a standardised aseptic manufacturing process, at the end of which, before the products are used therapeutically, strict quality and safety tests are conducted. Only if the specified quality criteria are met are the products then released for therapeutic application.

The efficacy of MukoCell has been shown in an open non-interventional study. However, to achieve market authorisation, the EMA requests that efficacy be further confirmed in a pivotal clinical study in direct comparison with native oral mucosa. This study will begin shortly and will involve a total of 200 patients, divided into two therapy groups of 100 patients each. Initial results of the study are expected by the beginning of 2023.

One goal of this clinical study is to show equivalence of the tissue-engineered product with native oral mucosa in urethral stricture treatment; the other goal is to clearly demonstrate the superiority of MukoCell over native oral mucosa as a graft, in terms of the aforementioned frequent and severe intraoral complications and impact on quality of life for patients.

The demonstration of MukoCells superiority is not only important regarding market authorisation, but also with respect to reimbursement by health insurances. The transplantation of native oral mucosa is a procedure developed by hospital surgeons. A critical examination of its safety and effectiveness has never been carried out, and complications are accepted if there is no alternative treatment.

Moreover, besides the surgical procedure which is paid for by the health insurance companies there are no additional costs associated with using native oral mucosa. In contrast, to justify additional costs arising from the use of a cultivated transplant, the efficacy, safety and superiority to native oral mucosa need to be proven.

Therefore, in the clinical trial, it is particularly important that the complications arising from excision of the transplant are recorded and documented as objectively as possible. Since the goal of surgery is to reconstruct the urethra, urologists pay little attention to intraoral complications and commonly play down their severity and importance.

Although the production of MukoCell is very complex and absolute sterility must be maintained during the three-week cultivation period, the costs are acceptable at several thousand euros. What pushes the costs even higher is the need to fulfil the requirements of the EMA in order to obtain marketing authorisation for the product: the planned clinical trial alone will cost around 10m. These costs must also be considered when pricing MukoCell.

The requirements of the regulatory authorities and health insurance companies not only influence the price of the products but also their availability. MukoCell has been on the market since 2013, but its approval is limited to Germany and only applies in a few individual cases due to the issue of reimbursement.

In a 2019 review the opinion was expressed that, due to the specificity of tissue-engineered products and the health benefits they offer, it would be advantageous to reconsider their regulatory requirements. The simplification of these requirements would allow the acceleration of these products into the market, faster availability for the patients and a decrease in the associated costs, making reimbursement less challenging for public health insurances in different countries.

Further, it was stated that the use of MukoCell represents a real, safe and efficient opportunity for patients with urethral stricture diseases. However, at present, regulatory, legal and financial issues represent important factors that restrict and slow down the wider use of MukoCell.

Soeren Liebig, CEOMukoCell GmbHBioMedizinzentrumDortmund+49 (0)23197426370s.liebig@mukocell.comwww.mukocell.com

Please note, this article will appear in issue 12 of Health Europa Quarterly, which will be available to read in February 2020.

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A milestone in the treatment of men's disease with regenerative medicine - Health Europa

The Alliance for Regenerative Medicine Outlines Recommendations on Enabling Cross-border and Regional Access to Advanced Therapy Medicinal Products (ATMPs) in Europe

Establish an ATMP coordination body at EU/EEA level

Ensure authorities in regions of treatment are compensated for costs of treating patients from other regions

Encourage greater alignment within Europe on product value assessment activities

BRUSSELS, BELGIUM 27 January, 2020

The Alliance for Regenerative Medicine (ARM), the international advocacy organization representing the cell and gene therapy and broader advanced therapies sector, today published a positioning paper outlining recommendations for the timely and effective access to cross-border healthcare for patients.

Todays new position paper focuses, and further elaborates, on the recommendations of ARMs July 2019 report on ensuring timely access to ATMPs in Europe (see the report here). It represents the views of the ARM members and aims to stimulate debate and reach consensus among key stakeholders, including marketing authorisation holders, payers and treatment centres, on solutions to ensure all European patients can secure access to ATMPs, irrespective of their country or region of origin.

Challenges to expanded ATMP access in Europe

Not all approved ATMPs are expected to be made available to all countries in Europe or to all regions of a given country.

The legal frameworks that grant the right to cross-border healthcare for patients in Europe are not optimal for ATMP treatments.

The lack of Health Technology Assessment (HTA)/pricing assessment in the patients country of origin and regional budgets or multiple payers/insurers in some countries can constitute barriers to cross-border or cross-region treatment with ATMPs.

ARMs key recommendations

In order to ensure that patients across Europe can access ATMPs, ARM recommends the following:

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Establish a one-stop shop ATMP coordination body at EU/EEA level to act as a broker between the different stakeholders and facilitate cross-border patient treatment and funding.

Create one-stop shop ATMP coordination bodies in countries with regional funding or with multiple payers/insurers to ensure authorities in the regions of treatment are compensated for the costs of treating patients from other regions.

Encourage more effective coordination of HTA activities to ensure greater alignment within Europe on product value assessment measures.

Additional recommended measures to facilitate industry engagement in existing initiatives could include: improved opportunities for cross-country collaboration, removing duplicative processes at national level, and adopting policy principles to enhance cross-country collaboration.

Janet Lambert, CEO of ARM, commented: Europe has always been a leader in ATMP innovation, both in R&D and getting products to market, however, to ensure that patients have access to these transformative treatments, there are several challenges that need to be overcome at EU, national and regional levels. This paper builds on the EU Market Access Report published in 2019 and the subsequent European stakeholder meeting in Brussels, and outlines the challenges and the recommendations that we, alongside our members, believe will most effectively get these therapies to patients in a sustainable manner.

To read the report in full, please follow this link.

Press inquiriesFor more information about the report or media requests, please contact Consilium Strategic Communications at arm@consilium-comms.com.

About the Alliance for Regenerative Medicine

The Alliance for Regenerative Medicine (ARM) is an international multi-stakeholder advocacy organization that promotes legislative, regulatory, and reimbursement initiatives necessary to facilitate access to life-giving advances in regenerative medicine worldwide. Founded in 2009, ARM works to increase public understanding of the field and its potential to transform human healthcare, providing business development and investor outreach services to support the growth of its 350+ member organizations worldwide. ARM represents the interests of therapeutic developers, academic research institutions, major medical centers, investors, and patient groups that comprise the broader regenerative medicine community and is the prominent international advocacy organization in this field.

ARM has 70+ members across 15 countries in Europe. ARM aims to work closely with European stakeholders, leveraging its membership to create a supportive commercial and regulatory environment to create better conditions for the development and commercialization of ATMPs in Europe; develop strong stakeholder support around proposed solutions to improve patient access to ATMPs; promote clear, predictable and efficient regulatory framework across Europe; and promote international convergence of key regulations and guidance. For more information, visit alliancerm.org.

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The Alliance for Regenerative Medicine Outlines Recommendations on Enabling Cross-border and Regional Access to Advanced Therapy Medicinal Products...

LAS VEGAS, Jan. 22, 2020 (GLOBE NEWSWIRE) -- Zhittya Genesis Medicine, Inc. (a private company) (Zhittya or the Company), has signed a $151.5 million USD international marketing partnership agreement with Regenerative Medicine of Latin America, Inc. for the exclusive rights to market and sell all biological drugs developed by Zhittya during a 30-year time period. The payments include an initial upfront payment valued at $76.5 million with the additional $75 million to be amortized through future milestone payments.

Zhittya is developing a family of biological drugs to treat diseases which are characterized by diminished blood flow, or perfusion, to specific tissues or organs. The diseases Zhittyas drugs are intended to treat address a variety of disorders and diseases, including: coronary heart disease, diabetic foot ulcers, stroke recovery, Parkinsons disease (PD), Alzheimers disease, and 14 additional major medical disorders characterized by insufficient blood perfusion.

According to the American Heart Associations 2019 Statistics at a Glance, heart disease is responsible for the death of approximately one out of three U.S. adults. In a U.S. Food and Drug Administration (FDA) clinical trial, Zhittyas heart-specific drug treatment demonstrated a successful triggering of new blood vessel growth in a diseased heart. According to a 2017 report by the World Health Organization, there are an estimated 20 million people who suffer from heart disorders in Latin America alone, 80% of which suffer from a particularly notorious form called small vessel disease, a disease that only Zhittyas drug has been able to address; the standard forms of treatment for coronary artery disease, including bypass and stenting procedures, can only be performed on larger arteries.

Our portfolio of drugs seeks to address diseases which directly cause the suffering and even death of over 50% of all adults, said Zhittya CEO Daniel C. Montano. In addition to the territories covered by our existing partnerships in North America, Europe and China, Latin America is particularly impacted by heart disease due to a variety of health and environmental concerns in the region. This agreement with Regenerative Medicine of Latin America is another major step forward to treating heart disease in Latin America and globally. Going forward, we believe we are on the path to a number of other major medical breakthroughs to address even more diseases caused by a lack of blood perfusion.

Dr. Jack Jacobs, President of Zhittya Genesis Medicine, stated, Our drug currently being developed to treat Parkinsons disease has demonstrated encouraging results with impressive outcomes in preclinical models of Parkinsons disease in rodents and primates. This drug has the potential to be a disease modifying agent; in preclinical studies it was shown to reverse the decline and actually stimulate the regeneration of dopamine-producing neurons, the root cause of Parkinsons disease in patients. According to a recent report from the Cleveland Clinic, the incidence rate of Parkinsons disease per 100,000 people was highest in Hispanics. We believe our drug can have a tremendous impact in this region in addition to our existing partnerships both domestically and internationally.

Dr. Jacobs added, We have filed applications and are advancing through the approval process to initiate Phase I clinical trials in Mexico for Parkinsons disease. We are also pursuing a second medical indication for patients with amyotrophic lateral sclerosis (ALS). It is our goal to be in a position to begin dosing patients with Parkinsons disease and ALS by early 2020, which should enable us to learn if our drug has the same beneficial effects in humans as it demonstrated in animals. These clinical trials that will hopefully begin very soon in Mexico will drive intense attention and interest to Regenerative Medicine of Latin America.

About Zhittya Genesis MedicineZhittya Genesis Medicine, Inc. is advancing a group of drugs which trigger the human bodys natural regeneration process. Our medicine initiates a biological response in the human body referred to as therapeutic angiogenesis, which will only occur in diseased tissues that become ischemic due to a lack of blood flow. In those areas with insufficient blood flow, the drug stimulates growth of new blood vessels, providing nourishment and removing metabolic waste products, thereby re-establishing normal cellular functions. Heart disease, stroke, peripheral artery disease (PAD) and diabetic foot ulcers are just some of the disorders the drugs can treat. Currently, over 75 human diseases are known to be caused by lack of blood flow to a tissue or organ. The Companys management has been working to advance its proprietary medicines for over 21 years and has expended in excess of $140 million USD to date in support of these efforts. To learn more, please visitzhittyaregenerativemedicine.com

About Regenerative Medicine of Latin AmericaRegenerative Medicine of Latin America, Inc. owns the 30-year exclusive rights to market and sell all drugs developed by Zhittya for the territories of Mexico and all Latin American countries south of Mexico. These areas encompass a population of over 600 million people. In addition to its vast population, Latin America also has some of the worlds highest rates of diabetes, heart disease, strokes and other diseases brought on by vascular dysfunction.

Zhittya Contact:

Daniel C. Montano, CEOZhittya Genesis Medicine, Inc.702-790-9980dan@zhittyamedicine.com

Investor Relations Contact:

Matt Glover and Tom ColtonGateway Investor Relations949-574-3860zhittya@gatewayir.com

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Zhittya Genesis Medicine Signs $151.5 Million Biopharmaceutical Marketing Partnership Agreement with Regenerative Medicine of Latin America - BioSpace

MIAMI, Jan. 21, 2020 (GLOBE NEWSWIRE) -- Organicell Regenerative Medicine Inc. (BPSR) (the Company) is pleased to provide shareholders and the investment community with an update on operations since its filing on November 1, 2018 of the Companys Annual Report on Form filing of Form 10-K for the year ended October 31, 2017, as well as the status of becoming fully compliant with SEC reporting obligations.

The Company is diligently working to complete its Quarterly Reports on Form 10-Q for the quarters ended January 31, 2018, April 30, 2018 and July 31, 2018 and its Annual Report on Form 10-K for the year ended October 31, 2018. In August 2019, the Company engaged Marcum LLP as its independent registered public accounting firm. The Company expects these reports to be completed and filed during the first calendar quarter of 2020. Following completion and filing of these reports, the Company expects to promptly proceed to preparation and filing of its Quarterly and Annual Reports for the fiscal year ended October 31, 2019, with the objective of becoming current in its SEC reporting requirements as soon as possible.

Since November 2018, the Company has remained focused on research and development activities and sale and distribution of anti-aging and cellular therapy derived products.

In February 2019, the Company recommenced its efforts to once again operate a perinatal tissue bank processing laboratory in Miami, Florida for the purpose of performing research and development and the manufacturing and processing of anti-aging and cellular therapy derived products. This new laboratory facility became operational in May 2019 and during the same period, the Company began producing products that are now being sold and distributed to its customers.

In addition, the Company has created what it believes is a world class research, medical and scientific advisory team. We believe that our team is one of the most qualified and industry reputable teams assembled to adequately address the current and expected future medical and regulatory challenges facing the Company and overall industry and to provide leadership in the ongoing development of superior quality products for use in the health care industry.

The Company has actively taken steps to assure that it meets compliance with current and anticipated United States Food and Drug Administration (FDA) regulations expected to be enforced beginning in November 2020 requiring that the sale of products that fall under Section 351 of the Public Health Services Act pertaining to marketing traditional biologics and human cells, tissues and cellular and tissue based products (HCT/Ps) can only be sold pursuant to an approved biologics license application (BLA). On July 14, 2019, the Company received Institutional Review Board (IRB) approval to proceed with two pilot studies in connection with the Companys efforts to obtain Investigation New Drug (IND) approval from the FDA and commence clinical trials in connection with the use of the Companys products and related treatment protocols for specific indications. The Company is aggressively pursuing efforts to obtain the aforementioned IND approvals and commence and complete those clinical studies as well as obtaining approval to commence additional studies for other specific indications it has identified that the use of its products will provide more favorable and desired health related benefits for patients seeking alternative treatment options than are currently available.

In an effort to increase sales and mitigate anticipated near future restrictions expected to be imposed by the FDA with respect to the use and distribution of Section 351 designated biologics, the Company is seeking to develop sales and distribution channels outside of the United States. In addition, the Company is focusing its efforts on developing other leading edge product offerings that would not fall within the FDA regulations for requiring a BLA license for U.S. manufacture and sale.

As a result of the Companys expected future increase in processing requirements and to enable it to perform certain advanced research and development activities, the Company is currently in negotiations to relocate its laboratory facility during the second calendar quarter of 2020 to a larger ISO 7 classified research and development and processing facility.

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The Company has also been actively developing and expanding its sales, marketing and distribution network which it believes that based on the quality of the Companys existing products, the Companys commitment to regulatory compliance and superior research and development resources, the Company believes that it will be able to achieve desired growth during 2020.

The Company expects to provide periodic updates on operational and financial reporting developments as warranted.

For more information regarding the Company please visit our website at http://www.organicell.com.

About Organicell Regenerative Medicine, Inc.

Organicell is a leading, fully integrated Company focused in the field of regenerative medicine. Our world class research, technology, manufacturing and clinical development team is focused on creating new biologic medicines to revolutionize the field of regenerative medicine. We believe that our ground-breaking research in the field of nanotechnology, specifically exosome enrichments and other micro vesicles, is the next frontier of stem cell-based therapeutics. Organicell is committed to creating life changing and lifesaving therapies for patients.

Our mission is to transform regenerative medicine by continuing to combine exosome technology with other synergistic therapies and become the healthcare technology incubator for biologic medicine.

CAUTIONARY COMMENT REGARDING FORWARD-LOOKING STATEMENTS

The foregoing contains "forward-looking statements" within the meaning of the Private Securities Litigation Reform Act of 1995. We intend for these forward-looking statements to be covered by the safe harbor provisions of the federal securities laws relating to forward-looking statements. This release contains forward-looking statements that reflect Organicell Regenerative Medicine Inc., and its subsidiaries, plans and expectations, financial situation, the ability to retain key personnel, product acceptance, the commercial success of any new products or technologies, success of clinical programs, ability to retain key customers, ability to expand sales and channels, and legislation or regulations affecting our operations and the ability to protect our patents and other intellectual property both domestically and internationally and other known and unknown risks and uncertainties. You are cautioned not to rely on these forward-looking statements. In this press release and related comments by Company management, words like "expect," "anticipate," "estimate," "intend", believes and similar expressions are used to identify forward-looking statements, representing management's current judgment and expectations about possible future events.

Management believes these forward-looking statements and the judgments upon which they are based to be reasonable, but they are not guarantees of future performance and involve numerous known and unknown risks, uncertainties and other factors that may cause the Company's actual results, performance, achievements or financial position to be materially different from any expressed or implied by these forward-looking statements. Important factors that could cause actual results to differ materially from the forward-looking statements are set forth in our Form 10-K and other filings with the SEC. Other information can be obtained at http://www.organicell.com. The contents of the Companys website are not incorporated by reference in this Press Release.

Specific information included in this press release may change over time and may or may not be accurate after the date of the release. Organicell has no intention and specifically disclaims any duty to update the information in this press releases.

CONTACT:Organicell Regenerative Medicine Inc.4045 Sheridan Ave.Suite 239Miami Beach, FL 33140Website:http: http://www.organicell.comPhone: (888) 963-7881Email: info@organicell.com

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Organicell Regenerative Medicine Inc. Provides Update On Operations and Financial Reporting Status - Yahoo Finance

New Delhi [India], Jan 23 (ANI/Business Wire India): On Saturday, January 18 2020, the Advancells Group and the International Fertility Center together ended their first workshop - Sub-Specialty Training in Application of Regenerative Medicine (STAR 2020).

The three-day workshop had specialized doctors, medical practitioners, learned scientists of Advancells, the leaders in cell manufacturing and processes and IFC, one of India's most prestigious Fertility institutes who were joined by candidates with MBBS/BAMS/BHMS/BPharma and Master's degree in Life Sciences.

The key-note speaker of the workshop was Dr Rita Bakshi, founder and chairperson of International Fertility Centre, the oldest fertility clinic and one of the most renowned IVF clinics in India, one of the organizers of the event.

Participants also had a privilege to listen to Dr Sachin Kadam, CTO, Advancells and gain hands-on experience in the preparation of PRP; Liposuction method; and Bone Marrow aspiration. All these techniques were talked about at length and demonstrated in the form of manual and kit-based models to help the candidates gain exposure.

Dr Punit Prabha, Head of Clinical Research and Dr Shradha Singh Gautam, Head of Lab Operations at Advancells successfully set the base of stem cell biology for the participants who were experts in gynecology field, stem cell research and pain specialist.

With the help of detailed analysis of 'Application of PRP for Skin rejuvenation'; 'Preparation of Micro-fragmented Adipose Tissue and Nano Fat & SVF (Stromal Vascular Fraction) from Adipose Tissue'; and 'Cell Culturing and Expansion in a Laboratory', applicants understood the application of stem cells in aesthetics, cosmetology, and anti-ageing.

"Educating young scientists about stem cells is important for us. With this workshop, we wanted to discuss and share the challenges and lessons we have learned in our journey of curing our customers," said Vipul Jain, founder and CEO of Advancells Group.

"We wanted to establish a more concrete knowledge base in the presence of subject matter experts and help our attendees in more possible ways. We are hopeful to have successfully achieved what we claimed with this workshop," he added.

Given the resounding success of the Sub-Specialty Training in Application of Regenerative Medicine (STAR 2020), it's hoped that the future events shall offer even greater wisdom to the participants by helping them improve and the lead the community into the age of greater awareness.

This story is provided by BusinessWire India. ANI will not be responsible in any way for the content of this article. (ANI/BusinessWire India)

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Advancells Group, IFC concluded their three-day workshop on Regenerative Medicine - Yahoo India News

Transaction Accelerates Anikas Revenue Growth, Broadens Joint Preservation and Restoration Product Portfolio, Enhances Commercial Capabilities and Expands Pipeline

BEDFORD, Mass., Jan. 24, 2020 (GLOBE NEWSWIRE) -- Anika Therapeutics, Inc.(ANIK), a global, integrated joint preservation and regenerative therapies company with products leveraging its proprietaryhyaluronic acid (HA) technology platform, today announced it has closed its acquisition of Parcus Medical, a leading, privately held sports medicine company.

Under the previously disclosed terms of the agreement, Anika acquired all outstanding membership interests of Parcus Medical in exchange for an upfront payment of approximately$35 millionin cash from the companys existing balance sheet, subject to customary closing adjustments. Parcus Medical unitholders will be eligible to receive an additional$60 millioncontingent upon the achievement of certain commercial milestones.

I want to congratulate our team on closing the Parcus Medical transaction and officially welcome the Parcus Medical team to the Anika family, said Joseph Darling, President and Chief Executive Officer of Anika Therapeutics. This acquisition immediately adds a diverse base of high-growth revenue and will help us achieve the objectives we set forth in our five-year strategic plan. We can now turn our attention to executing our integration plan and continuing to transform Anika into a leading global sports and regenerative medicine company.

Parcus Medical has a diverse product family that helps facilitate surgical procedures on the shoulder, knee, hip and distal extremities. The acquisition significantly expands Anikas offerings into the fast-growing ambulatory surgical center market. The Parcus Medical executive team, led by PresidentMark Brunsvold, will join Anika and continue to lead the Parcus Medical business.

SVB Leerink LLCacted as exclusive financial advisor to Anika andSullivan & Cromwell LLPacted as Anikas legal counsel in connection with the Parcus Medical transaction.

AboutAnika Therapeutics, Inc.Anika Therapeutics, Inc.(ANIK) is a global, integrated joint preservation and regenerative therapies company based inBedford, Mass.Anika is committed to delivering therapies to improve the lives of patients across a continuum of care from osteoarthritis pain management to joint preservation and restoration. The company has more than two decades of global expertise commercializing more than 20 products based on its proprietaryhyaluronic acid (HA) technology platform. For more information about Anika, please visitwww.anikatherapeutics.com.

Forward-Looking StatementsThis press release includes forward-looking statements within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934, as amended, concerning, but not limited to, the acquisition of Parcus Medical and the effects of the acquisition.The Securities and Exchange Commission("SEC") encourages companies to disclose forward-looking statements so that investors can better understand a companys future prospects and make informed investment decisions. Forward-looking statements are subject to risks and uncertainties, many of which are outside our control, which could cause actual results to differ materially from these statements. Therefore, you should not rely on any of these forward-looking statements. Forward-looking statements can be identified by such words as "will," "likely," "may," "believe," "expect," "anticipate," "intend," "seek," "designed," "develop," "would," "future," "can," "could," and other expressions that are predictions of or indicate future events and trends and that do not relate to historical matters. All statements other than statements of historical facts included in this press release regarding our strategies, prospects, financial condition, operations, costs, plans, and objectives are forward-looking statements.

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Anika Therapeutics Closes Acquisition of Parcus Medical - Yahoo Finance

A thorough study of the competitive landscape of the Industrial Tripods Market has been given, presenting insights into the company profiles, financial status, recent developments, mergers and acquisitions, and the SWOT analysis. This research report will give a clear idea to readers about the overall market scenario to further decide on this market projects.

The report analysis the leading players of the Industrial Tripods Market by inspecting their market share, recent developments, new product launches, partnerships, mergers, or acquisitions, and their target markets. This report also includes an exhaustive analysis of their product profiles to explore the products and applications their operations are concentrated on in the Industrial Tripods Market. Additionally, the report gives two distinct market forecasts, one from the perspective of the producer and another from that of the consumer. It also offers valuable recommendations for new as well as established players of the Industrial Tripods Market. It also provides beneficial insights for both new as well as established players of the Industrial Tripods Market.

ThisPress Release will help you to understand the Volume, growth with Impacting Trends. Click HERE To get SAMPLE PDF (Including Full TOC, Table & Figures) athttps://www.persistencemarketresearch.co/samples/23425

This report provides detailed historical analysis of global market for Industrial Tripods from 2014-2018, and provides extensive market forecasts from 2018 2028 by region country and subsectors. It covers the sales volume, price, revenue, gross margin, historical growth and future perspectives in the Industrial Tripods Market.

Overview:

The next section offers an overview of the Industrial Tripods Market. This section includes definition of the product Industrial Tripods , along with insights on dynamics contributing towards growth of the market. The overview also throws light on year-on-year growth and market value defining the future progress and decline of the global Industrial Tripods . Statistics on the year-on-year growth provides readers with a broader view on expected progress patterns reshaping growth over the forecast period 2018 2028.

In the succeeding section, the report offers insights on major trends, retrains and drivers from demand, supply and macro-economic perspectives. The report also focuses on impact analysis of key drivers and restraints that offers better decision-making insights to clients.

The report further provides the readers with information on the leading technology and advancements traced in the Industrial Tripods Market. Up-to-date information and latest advancements regarding growth opportunities can benefit the leading manufacturers of Industrial Tripods . With continuous evolution and advancements in technology, tracking the latest trends and developments is fundamental for Industrial Tripods manufacturers to formulate key business strategies. Detailed insights regarding the supply chain, list of distributors, raw material sourcing, cost structure, and pricing analysis are provided in this section.

Considering the Industrial Tripods Markets wide scope, PMRs report provides in-depth insights & forecast based on segment-wise analysis. The Industrial Tripods Market has been categorized on the basis of middleware type, sector, deployment type, and region. This sections delivers a comprehensive segmentation analysis, along with a detailed country-wise forecast offered on all parameters.

In the last section, the report provides information regarding the competitive landscape, along with a dashboard view of the market players and company analysis. This competitive intelligence is based on the providers categories across the value chain, and their presence in the Industrial Tripods Market.

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Research Methodology

PMR is committed to offer unbiased and independent market research solutions to its clients. Each market report of PMR is compiled after months of exhaustive research. We bank on a mix of tried-and-tested and innovative research methodologies to offer the most comprehensive and accurate information. Our main sources of research include,

The Industrial Tripods Market research is carried out at the different stages of the business lifecycle from the production of a product, cost, launch, application, consumption volume and sale. The research offers valuable insights into the marketplace from the beginning including some sound business plans chalked out by prominent market leaders to establish a strong foothold and expand their products into one thats better than others.

We provide detailed product mapping and investigation of various market scenarios. Our expert analysts provide a thorough analysis and breakdown of the market presence of key market leaders. We strive to stay updated with the recent developments and follow the latest company news related to the industry players operating in the Industrial Tripods Market. This helps us to comprehensively analysis the individual standing of the companies as well as the competitive landscape. Our vendor landscape analysis offers a complete study to help you gain the upper hand in the competition.

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Table of Contents

Report Overview: It includes six chapters, viz. research scope, major manufacturers covered, market segments by type, Industrial Tripods Market segments by application, study objectives, and years considered.

Global Growth Trends: There are three chapters included in this section, i.e. industry trends, the growth rate of key producers, and production analysis.

Industrial Tripods Market Share by Manufacturer: Here, production, revenue, and price analysis by the manufacturer are included along with other chapters such as expansion plans and merger and acquisition, products offered by key manufacturers, and areas served and headquarters distribution.

Market Size by Type: It includes analysis of price, production value market share, and production market share by type.

Market Size by Application: This section includes Industrial Tripods Market consumption analysis by application.

Profiles of Manufacturers: Here, leading players of the Industrial Tripods market are studied based on sales area, key products, gross margin, revenue, price, and production.

Industrial Tripods Market Value Chain and Sales Channel Analysis: It includes customer, distributor, Industrial Tripods Market value chain, and sales channel analysis.

Market Forecast Production Side: In this part of the report, the authors have focused on production and production value forecast, key producers forecast, and production and production value forecast by type.

About us:

PMR is a third-platform research firm. Our research model is a unique collaboration of data analytics and market research methodology to help businesses achieve optimal performance.

To support companies in overcoming complex business challenges, we follow a multi-disciplinary approach. At PMR, we unite various data streams from multi-dimensional sources. By deploying real-time data collection, big data, and customer experience analytics, we deliver business intelligence for organizations of all sizes.

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Regenerative Medicine Adoption of Innovative Offerings and Forecast 2019-2026 - Melanian News

Agreements Contingent on FTC and European Commission Approval and Completion of AbbVie's Pending Acquisition of Allergan

NORTH CHICAGO, Ill. and DUBLIN, Jan. 27, 2020 /PRNewswire/ -- AbbVie (NYSE: ABBV), a research-based global biopharmaceutical company, and Allergan (NYSE: AGN), a leading global pharmaceutical company, today announced that Allergan has entered into definitive agreements to divest brazikumab (IL-23 inhibitor) and Zenpep (pancrelipase). These agreements are in conjunction with the ongoing regulatory approval process for AbbVie's acquisition of Allergan.

AstraZeneca (NYSE: AZN) will acquire brazikumab, an investigational IL-23 inhibitor in Phase 2b/3 development for Crohn's Disease and in Phase 2 development for ulcerative colitis, including global development and commercial rights.

Nestle (Swiss: NESN) will acquire and take full operational ownership of Zenpep upon closing the transaction with customary transition support from Allergan. Zenpep is a treatment, which is available in the United States, for exocrine pancreatic insufficiency due to cystic fibrosis and other conditions. Nestle also will be acquiring Viokace, another pancreatic enzyme preparation, as part of the same transaction.

"These definitive agreements represent significant progress toward the completion of our acquisition of Allergan," said Richard A. Gonzalez, chairman and chief executive officer, AbbVie. "The new combined organization will be well positioned to deliver on our mission to patients with a broad portfolio of innovative therapies."

"Today, we move another step closer to completing AbbVie's acquisition of Allergan. Allergan's commercial and R&D teams have invested so much of themselves into the development of brazikumab and the commercialization of Zenpep, and these divestiture agreements will enable that work to continue following the close of our planned acquisition," said Brent Saunders, chairman and chief executive officer of Allergan.

The closings of the acquisitions of brazikumab and Zenpep are contingent upon receipt of U.S. Federal Trade Commission and European Commission approval, closing of AbbVie's pending acquisition of Allergan and the satisfaction of other customary closing conditions.

On January 10, 2020, AbbVie and Allergan received conditional approval of the transaction by the European Commission, subject to the approved divestiture of brazikumab and other conditions.

AbbVie and Allergan continue to expect a first-quarter 2020 close of their pending transaction, subject to receipt of required regulatory approvals and other closing conditions.

About AbbVieAbbVie is a global, research-driven biopharmaceutical company committed to developing innovative advanced therapies for some of the world's most complex and critical conditions. The company's mission is to use its expertise, dedicated people and unique approach to innovation to markedly improve treatments across four primary therapeutic areas: immunology, oncology, virology and neuroscience. In more than 75 countries, AbbVie employees are working every day to advance health solutions for people around the world. For more information about AbbVie, please visit us at http://www.abbvie.com. Follow @abbvie on Twitter or view careers on our Facebook or LinkedIn page.

About Allergan plcAllergan plc (NYSE: AGN), headquartered in Dublin, Ireland, is a global pharmaceutical leader focused on developing, manufacturing and commercializing branded pharmaceutical, device, biologic, surgical and regenerative medicine products for patients around the world. Allergan markets a portfolio of leading brands and best-in-class products primarily focused on four key therapeutic areas including medical aesthetics, eye care, central nervous system and gastroenterology. As part of its approach to delivering innovation for better patient care, Allergan has built one of the broadest pharmaceutical and device research and development pipelines in the industry.

With colleagues and commercial operations located in approximately 100 countries, Allergan is committed to working with physicians, healthcare providers, and patients to deliver innovative and meaningful treatments that help people around the world live longer, healthier lives every day.

For more information, visit Allergan's website at http://www.Allergan.com.

About ZenpepZENPEP (pancrelipase) is a prescription medication for people who cannot digest food normally because their pancreas does not make enough enzymes. ZENPEP may help your body use fats, proteins, and sugars from food. ZENPEP contains a mixture of digestive enzymes (lipases, proteases, and amylases) from pig pancreas. In clinical studies, individuals with exocrine pancreatic insufficiency associated with cystic fibrosis absorbed more fat from foods than those treated with a placebo.

About BrazikumabBrazikumab is a monoclonal antibody that binds to the IL23 receptor and is in development for Crohn's Disease and Ulcerative Colitis with a companion biomarker. Brazikumab selectively blocks the IL23 immune signal, preventing intestinal inflammation. The Phase IIb/III INTREPID program is underway to assess brazikumab compared to placebo or adalimumab in Crohn's Disease. The Phase II EXPEDITION trial is underway to assess brazikumab compared to placebo or vedolizumab in Ulcerative Colitis.

Forward-Looking StatementsThis announcement contains certain forward-looking statements, including with respect to the pending acquisition involving AbbVie and Allergan, Allergan's divestitures of brazikumab and Zenpep and AbbVie's, Allergan's and/or the combined group's estimated or anticipated future business, performance and results of operations and financial condition, including estimates, forecasts, targets and plans for AbbVie and, following the acquisition, if completed, the combined group. The words "believe," "expect," "anticipate," "project" and similar expressions, among others, generally identify forward-looking statements. These forward-looking statements are subject to risks and uncertainties that may cause actual results to differ materially from those indicated in the forward-looking statements. Such risks and uncertainties include, but are not limited to, the possibility that the divestitures and/or the pending acquisition will not be pursued, failure to obtain necessary regulatory approvals or required financing or to satisfy any of the other conditions to the pending acquisition, adverse effects on the market price of AbbVie's shares of common stock or Allergan's ordinary shares and on AbbVie's or Allergan's operating results because of a failure to complete the pending acquisition, failure to realize the expected benefits of the pending acquisition, failure to promptly and effectively integrate Allergan's businesses, negative effects relating to the announcement of the pending acquisition or any further announcements relating to the pending acquisition or the consummation of the pending acquisition on the market price of AbbVie's shares of common stock or Allergan's ordinary shares, significant transaction costs and/or unknown or inestimable liabilities, potential litigation associated with the pending acquisition, general economic and business conditions that affect the combined companies following the consummation of the pending acquisition, changes in global, political, economic, business, competitive, market and regulatory forces, future exchange and interest rates, changes in tax laws, regulations, rates and policies, future business acquisitions or disposals and competitive developments. These forward-looking statements are based on numerous assumptions and assessments made in light of AbbVie's or, as the case may be, Allergan's experience and perception of historical trends, current conditions, business strategies, operating environment, future developments and other factors it believes appropriate. By their nature, forward-looking statements involve known and unknown risks and uncertainties because they relate to events and depend on circumstances that will occur in the future. The factors described in the context of such forward-looking statements in this announcement could cause AbbVie's plans with respect to Allergan or AbbVie's or Allergan's actual results, performance or achievements, industry results and developments to differ materially from those expressed in or implied by such forward-looking statements. Although it is believed that the expectations reflected in such forward-looking statements are reasonable, no assurance can be given that such expectations will prove to have been correct and persons reading this announcement are therefore cautioned not to place undue reliance on these forward-looking statements which speak only as of the date of this announcement. Additional information about economic, competitive, governmental, technological and other factors that may affect AbbVie or Allergan is set forth in AbbVie's and Allergan's periodic public filings with the U.S. Securities and Exchange Commission, including, but not limited to, AbbVie's and Allergan's Annual Report on Form 10-K for the year ended December 31, 2018, Quarterly Report on Form 10-Q for the quarterly period ended March 31, 2019, Quarterly Report on Form 10-Q for the quarterly period ended June 30, 2019, Quarterly Report on Form 10-Q for the quarterly period ended September 30, 2019 and, from time to time, AbbVie's and Allergan's other investor communications, in each case, the contents of which are not incorporated by reference into, nor do they form part of, this announcement.

Any forward-looking statements in this announcement are based upon information available to AbbVie, Allergan and/or their respective board of directors, as the case may be, as of the date of this announcement and, while believed to be true when made, may ultimately prove to be incorrect. Subject to any obligations under applicable law, none of AbbVie, Allergan or any member of their respective board of directors undertakes any obligation to update any forward-looking statement whether as a result of new information, future developments or otherwise, or to conform any forward-looking statement to actual results, future events, or to changes in expectations. All subsequent written and oral forward-looking statements attributable to AbbVie, Allergan or their respective board of directors or any person acting on behalf of any of them are expressly qualified in their entirety by this paragraph.

Statement Required by Irish Takeover RulesThe Directors of AbbVie Inc. accept responsibility for the information contained in this announcement. To the best of their knowledge and belief (having taken all reasonable care to ensure such is the case), the information contained in this announcement is in accordance with the facts and does not omit anything likely to affect the import of such information.

The Allergan directors accept responsibility for the information contained in this report. To the best of the knowledge and belief of the Allergan directors (who have taken all reasonable care to ensure such is the case), the information contained in this report for which they accept responsibility is in accordance with the facts and does not omit anything likely to affect the import of such information.

Any holder of 1% or more of any class of relevant securities of Allergan plc or AbbVie Inc. may have disclosure obligations under Rule 8.3 of the Irish Takeover Panel Act, 1997, Takeover Rules 2013.

View original content:http://www.prnewswire.com/news-releases/abbvie-and-allergan-announce-agreements-to-divest-brazikumab-and-zenpep-300993294.html

SOURCE AbbVie

Company Codes: NYSE:ABBV, NYSE:AGN, NYSE:AZN, Swiss:NESN, OTC-PINK:NSRGY

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AbbVie and Allergan Announce Agreements to Divest Brazikumab and Zenpep - BioSpace