Translating stem cell research into development of cellular drugs—a perspective from manufacture of stem cell products and CMC considerations

The development of human pluripotent stem cell (PSC)‐derived medicinal products has been gathering steam in recent years, but the translation of research protocols into GMP production remains a daunting task. The challenges not only reside with the nature of cellular therapeutics but are also rooted in the general inexperience in industry‐scale production of stem cell products. Manufacturers of PSC‐derived products should be aware of the technical nuances and take a holistic approach toward early planning and engagement with their academic partners. While not all issues will be readily resolved soon, the collective knowledge and consensus by the manufacturers and key stakeholders will help to guide rapid progression of the field.     

Stromal cell‐derived factor‐1/Exendin‐4 cotherapy facilitates the proliferation,migration and osteogenic differentiation of human periodontal ligament stem cells in vitro and promotes periodontal bone regeneration in vivo

ObjectivesStromal cell‐derived factor‐1 (SDF‐1) actively directs endogenous cell homing. Exendin‐4 (EX‐4) promotes stem cell osteogenic differentiation. Studies revealed that EX‐4 strengthened SDF‐1‐mediated stem cell migration. However, the effects of SDF‐1 and EX‐4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo.MethodsCell‐counting kit‐8 (CCK8), transwell assay, qRT‐PCR and western blot were used to determine the effects and mechanism of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF‐1 and systemic injection of EX‐4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo.ResultsSDF‐1/EX‐4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis‐related gene expression compared to SDF‐1 or EX‐4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF‐1/EX‐4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF‐1/EX‐4 cotherapy significantly promoted new bone formation, recruited more CXCR4⁺ cells and CD90⁺/CD34^‐ stromal cells to the defects, enhanced early‐stage osteoclastogenesis and osteogenesis‐related markers expression in regenerated bone compared to control, SDF‐1 or EX‐4 in vivo.ConclusionsSDF‐1/EX‐4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration.     

Development and regulation of stem cell‐based therapies in China

BackgroundClinical researches of stem cell‐based therapies are highly active in China, while it was arduous to determine the most effective way of clinical translation of those advanced therapies.MethodsThis article briefly introduced the regulatory framework development, the progress in stem cell clinical researches and clinical trials of commercially developed stem cell‐based products, as well as the clinical review concerns of stem cell‐based products in China.Main findingsThe current regulatory framework of stem cell clinical researches in China was launched in 2015, when regulatory authorities issued “Administrative Measures on Stem Cell Clinical Research” (AMSCCR) detailing the rules of stem cell clinical research. Thereafter, the rapidly growing stem cell clinical researches were rigorously managed and clinical use of stem cell therapy was halted. Meanwhile, commercially developed stem cell‐based products are supervised by Drug Administration Law (DAL).ConclusionThe regulatory framework of stem cell‐based therapy in China has progressed in the last few decades, which is currently regulated according to AMSCCR and DAL. Well‐designed and patient‐focused clinical trial is required for commercially developed stem cell‐based products, and definite clinical benefit evidence is crucial to obtain marketing authorization.

This article briefly introduced the regulatory framework development of stem cell‐based therapy, progress in stem cell‐based clinical studies and clinical review concerns in China.     

Msps governs acentrosomal microtubule assembly and reactivation of quiescent neural stem cells

The ability of stem cells to switch between quiescence and proliferation is crucial for tissue homeostasis and regeneration. Drosophila quiescent neural stem cells (NSCs) extend a primary cellular protrusion from the cell body prior to their reactivation. However, the structure and function of this protrusion are not well established. Here, we show that in the protrusion of quiescent NSCs, microtubules are predominantly acentrosomal and oriented plus‐end‐out toward the tip of the primary protrusion. We have identified Mini Spindles (Msps)/XMAP215 as a key microtubule regulator in quiescent NSCs that governs NSC reactivation via regulating acentrosomal microtubule growth and orientation. We show that quiescent NSCs form membrane contact with the neuropil and E‐cadherin, a cell adhesion molecule, localizes to these NSC‐neuropil junctions. Msps and a plus‐end directed motor protein Kinesin‐2 promote NSC cell cycle re‐entry and target E‐cadherin to NSC‐neuropil contact during NSC reactivation. Together, this work establishes acentrosomal microtubule organization in the primary protrusion of quiescent NSCs and the Msps‐Kinesin‐2 pathway that governs NSC reactivation, in part, by targeting E‐cad to NSC‐neuropil contact sites.     

Conditioned medium derived from 3D tooth germs: A novel cocktail for stem cell priming and early in vivo pulp regeneration

ObjectivesConditioned medium (CM) from 2D cell culture can mitigate the weakened regenerative capacity of the implanted stem cells. However, the capacity of 3D CM to prime dental pulp stem cells (DPSCs) for pulp regeneration and its protein profile are still elusive. We aim to investigate the protein profile of CM derived from 3D tooth germs, and to unveil its potential for DPSCs‐based pulp regeneration.Materials and MethodsWe prepared CM of 3D ex vivo cultured tooth germ organs (3D TGO‐CM) and CM of 2D cultured tooth germ cells (2D TGC‐CM) and applied them to prime DPSCs. Influences on cell behaviours and protein profiles of CMs were compared. In vivo pulp regeneration of CMs‐primed DPSCs was explored using a tooth root fragment model on nude mice.ResultsTGO‐CM enhanced DPSCs proliferation, migration, in vitro mineralization, odontogenic differentiation, and angiogenesis performances. The TGO‐CM group generated superior pulp structures, more odontogenic cells attachment, and enhanced vasculature at 4 weeks post‐surgery, compared with the TGC‐CM group. Secretome analysis revealed that TGO‐CM contained more odontogenic and angiogenic growth factors and fewer pro‐inflammatory cytokines. Mechanisms leading to the differential CM profiles may be attributed to the cytokine–cytokine receptor interaction and PI3K‐Akt signalling pathway.ConclusionsThe unique secretome profile of 3D TGO‐CM made it a successful priming cocktail to enhance DPSCs‐based early pulp regeneration.     

Manufacturing with pluripotent stem cells (PSConf 2021): Key issues for future research and development

Human pluripotent stem cells (hPSC) have the capability to deliver novel cell‐based medicines that could transform medical treatments for a wide range of diseases including age‐related degenerative disorders and traumatic injury. In spite of significant investment in this area, due to the novel nature of these hPSC‐based medicines, there are challenges in almost all aspects of their manufacturing including bioprocessing, characterization and delivery. The Chinese Academy of Sciences and the Chinese Society for Stem Cell Research have collaborated to create a new discussion forum called PSConf 2021 (Pluripotent Stem Cell Conference 2021), intended to promote exchanges in communication on cutting‐edge developments and international coordination in hPSC manufacturing. The PSConf 2021 addressed crucial topics in stem cell‐based manufacturing, including stem cell differentiation, culture scale‐up, product formulation and release. This report summarizes the proceedings and conclusions from the discussion sessions, and it is accompanied by publication of individual papers from the speakers at the PSConf 2021.Significance StatementThe PSConf 2021 meeting has brought together speakers and delegates from more than 20 countries in an informal discussion forum focusing on the manufacture of cell‐based medicines using hPSCs. The conference discussion sessions enabled an open exchange of information on the latest developments, ideas on key challenges and their potential solutions. It also captured the experiences and lessons learnt by professionals who had been in the field from the earliest applications of human embryonic stem cells, and presented a diverse range of new potential pluripotent stem cell‐based medicines that are now under development, with some already in clinical trials.

Challenges in the manufacture of human plurtipotent stem cell‐based medicines.     

Biobanking of human pluripotent stem cells in China

In recent years, significant progress has been made internationally in the development of human pluripotent stem cell (hPSC)‐derived products for serious and widespread disorders. Biobanking of the cellular starting materials is a crucial component in the delivery of safe and regulatory compliant cell therapies. In China, key players in these developments have been the recently launched National Stem Cell Resource Center (NSCRC) and its partner organizations in Guangzhou and Shanghai who together, have more than 600 hPSC lines formally recorded in the Chinese Ministry of Science and Technology''s stem cell registry. In addition, 47 of these hPSCs have also been registered with the hPSCreg project which means they are independently certified for use in European Commission funded research projects. The NSCRC are currently using their own cell lines to manufacture eight different cell types qualified for clinical use, that are being used in nine clinical studies for different indications. The Institute of Zoology at the Chinese Academy of Sciences (IOZ‐CAS) has worked with NSCRC to establish Chinese and international standards in stem cell research. IOZ‐CAS was also a founding partner in the International Stem Cell Banking Initiative which brings together key stem cell banks to agree minimum standards for the provision of pluripotent stem cells for research and clinical use. Here, we describe recent developments in China in the establishment of hPSCs for use in the manufacture of cell therapies and the significant national and international coordination which has now been established to promote the translation of Chinese hPSC‐based products into clinical use according to national and international standards.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells

ObjectivesDelivery systems that provide time and space control have a good application prospect in tissue regeneration applications, as they can effectively improve the process of wound healing and tissue repair. In our experiments, we constructed a novel micro‐RNA delivery system by linking framework nucleic acid nanomaterials to micro‐RNAs to promote osteogenic differentiation of mesenchymal stem cells.Materials and MethodsTo verify the successful preparation of tFNAs–miR‐26a, the size of tFNAs–miR‐26a were observed by non‐denaturing polyacrylamide gel electrophoresis and dynamic light scattering techniques. The expression of osteogenic differentiation‐related genes and proteins was investigated by confocal microscope, PCR and western blot to detect the impact of tFNAs–miR‐26a on ADSCs. And finally, Wnt/β‐catenin signaling pathway related proteins and genes were detected by confocal microscope, PCR and western blot to study the relevant mechanism.ResultsBy adding this novel complex, the osteogenic differentiation ability of mesenchymal stem cells was significantly improved, and the expression of alkaline phosphatase (ALP) on the surface of the cell membrane and the formation of calcium nodules in mesenchymal stem cells were significantly increased on days 7 and 14 of induction of osteogenic differentiation, respectively. Gene and protein expression levels of ALP (an early marker associated with osteogenic differentiation), RUNX2 (a metaphase marker), and OPN (a late marker) were significantly increased. We also studied the relevant mechanism of action and found that the novel nucleic acid complex promoted osteogenic differentiation of mesenchymal stem cells by activating the canonical Wnt signaling pathway.ConclusionsThis study may provide a new research direction for the application of novel nucleic acid nanomaterials in bone tissue regeneration.

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells.     

The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti‐apoptotic Bcl‐2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl‐2 antagonist ABT‐199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro‐apoptotic BH3‐only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl‐2 and the marked susceptibility to Bcl‐2 antagonism. In line with these observations, conditional knockout of Bcl‐2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl‐2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl‐2‐induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche‐instructive mechanism of Bcl‐2‐regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.     

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10‐R98S). Our data support that RPL10‐R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock‐in of RPL10‐R98S was associated with a 1.7‐ to 2.5‐fold increased production of four transiently expressed recombinant proteins and 1.7‐fold for one out of two stably expressed proteins. In CHO‐S cells, eGFP reached a 2‐fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10‐R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10‐R98S phenotypes can maximize its exploitability for the mammalian protein production industry.     

Analysis and pharmacological modulation of senescence in human epithelial stem cells

Human epithelial stem cells (ESCs) are characterized by long‐term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly‐ectodermal dysplasia‐clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ‐secretase inhibitor; N‐[N‐(3, 5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine T‐butyl ester). All cells were able to differentiate in vitro but exhibited variable self‐renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up‐ or down‐regulated during their lifetime, thus allowing to identify druggable gene networks and off‐the‐shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.     

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

ObjectivesSalivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration.Materials and MethodsWe used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases.ResultsIntact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration.ConclusionsOur findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.     

Meeting report: fourth international congress of the Society for Melanoma Research

The 4th international melanoma congress of the Society for Melanoma Research (SMR), organized by Marianne Berwick (University of New Mexico), Paul Chapman (Memorial Sloan-Kettering Cancer Center), Rene Gonzalez (University of Colorado) and Ze'ev Ronai (Burnham Institute), was held at the Marriott Hotel in downtown New York on November 2007. The congress was attended by a record high number of attendees (over 500 delegates) who joined to discuss recent advances in melanoma biology and therapy. About 40% of the participants arrived from 39 countries, a testament to the high impact of this annual gathering on the international melanoma community. Over 120 of the participants were students or postdoctoral fellows, representing a most impressive fraction of young scientists engaged in melanoma research. The meeting consisted of more than 50 plenary and minisymposia presentations, stimulating the exchange of unpublished data and novel ideas, and helping to forge new collaborations that are anticipated to facilitate significant advances in basic, translational and clinical melanoma research. Another major focus of this meeting was over 160 posters, which were heavily attended and provided an effective forum for extensive informal discussions. This report will highlight the major scientific themes and advances of this most successful meeting, and provide a useful perspective on the current state of melanoma research, as well as where the field should be heading.     

Amphiregulin promotes hair regeneration of skin‐derived precursors via the PI3K and MAPK pathways

ObjectivesThere are significant clinical challenges associated with alopecia treatment, including poor efficiency of related drugs and insufficient hair follicles (HFs) for transplantation. Skin‐derived precursors (SKPs) exhibit great potential as stem cell‐based therapies for hair regeneration; however, the proliferation and hair‐inducing capacity of SKPs gradually decrease during culturing.Materials and MethodsWe describe a 3D co‐culture system accompanied by kyoto encyclopaedia of genes and genomes and gene ontology enrichment analyses to determine the key factors and pathways that enhance SKP stemness and verified using alkaline phosphatase assays, Ki‐67 staining, HF reconstitution, Western blot and immunofluorescence staining. The upregulated genes were confirmed utilizing corresponding recombinant protein or small‐interfering RNA silencing in vitro, as well as the evaluation of telogen‐to‐anagen transition and HF reconstitution in vivo.ResultsThe 3D co‐culture system revealed that epidermal stem cells and adipose‐derived stem cells enhanced SKP proliferation and HF regeneration capacity by amphiregulin (AREG), with the promoted stemness allowing SKPs to gain an earlier telogen‐to‐anagen transition and high‐efficiency HF reconstitution. By contrast, inhibitors of the phosphoinositide 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathways downstream of AREG signalling resulted in diametrically opposite activities.ConclusionsBy exploiting a 3D co‐culture model, we determined that AREG promoted SKP stemness by enhancing both proliferation and hair‐inducing capacity through the PI3K and MAPK pathways. These findings suggest AREG therapy as a potentially promising approach for treating alopecia.     

Delineating the relationship between immune system aging and myogenesis in muscle repair

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y‐O) or old to old (O‐O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL‐10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y‐O chimeras). Improved immune responses in Y‐O chimeras were associated with greater satellite cell proliferation compared with O‐O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age‐related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.