Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells. METHODS: We used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency. RESULTS: Results indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells. CONCLUSIONS: These studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.

Release Assays and Potency Assays for CAR T-Cell Interventions.

Chimeric antigen receptor (CAR) T-cells are considered "living drugs" and offer a compelling alternative to conventional anticancer therapies. Briefly, T-cells are redirected, using gene engineering technology, toward a specific cancer cell surface target antigen via a synthetic chimeric antigen receptor (CAR) protein. CARs have a modular design comprising four main structures: an antigen-binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains for T-cell activation. A major challenge in the CAR T-cell manufacturing field is balancing product quality with scalability and cost-effectiveness, especially when transitioning from an academic clinical trial into a marketed product, to be implemented across many collection, manufacturing, and treatment sites. Achieving product consistency while circumnavigating the intrinsic variability associated with autologous products is an additional barrier. To overcome these limitations, a robust understanding of the product and its biological actions is crucial to establish a target product profile with a defined list of critical quality attributes to be assessed for each batch prior to product certification. Additional challenges arise as the field progresses, such as new safety considerations associated with the use of allogenic T-cells and genome editing tools. In this chapter, we will discuss the release and potency assays required for CAR T-cell manufacturing, covering their relevance, current challenges, and future perspectives.

Illustrative Potency Assay Examples from Approved Therapies.

Advanced therapy medicinal products (ATMP) encompass a new type of drugs resulting from the manipulation of genes, cells, and tissues to generate innovative medicinal entities with tailored pharmaceutical activity. Definition of suitable potency tests for product release are challenging in this context, in which the active ingredient is composed of living cells and the mechanism of action often is poorly understood. In this chapter, we present and discuss actual potency assays used for the release of representative commercial ATMP from each category of products (namely, KYMRIAH® (tisagenlecleucel), Holoclar® (limbal epithelial stem cells), and PROCHYMAL®/RYONCIL™ (remestemcel-L)). We also examine concerns related to the biological relevance of selected potency assays and challenges ahead for harmonization and broader implementation in compliance with current quality standards and regulatory guidelines.

Determinism and variability of the morphogenesis pathways.

Along with a strict determinism of early embryogenesis in most living organisms, some of them exhibit variability of cell fates and developmental pathways. Here we discuss the phenomena of determinism and variability of developmental pathways, defining its dependence upon cell potency, cell sensitivity to the external signals and cell signaling. We propose a set of conjectures on the phenomenon of variability of developmental pathways, and denote a difference between a normal (local) variability, leading to an invariant final structure (e.g., embryo shape), and fundamental one, which is a switching between different developmental pathways, leading to different possible structures. For illustrating our conjectures, we analyzed early developmental stages of plant embryos with different levels of variability of morphogenesis pathways, and provide a set of computational experiments by Morphogenesis Software.

De novo genesis of retinal ganglion cells by targeted expression of Klf4 in vivo.

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2 In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina.This article has an associated 'The people behind the papers' interview.

Mapping human serum-induced gene networks as a basis for the creation of biomimetic periosteum for bone repair.

BACKGROUND: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation. AIM: This study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment. METHODS: The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum. RESULTS: Master regulators of transcriptional networks were identified, and an optimized periosteum-derived growth factor cocktail (PD-GFC; containing ß-estradiol, FGF2, TNFα, TGFß, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regard to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a three-dimensional collagen type 1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a down-regulated WNT and TGFß signature and an up-regulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature. CONCLUSION: This study highlights the first stage in the development of a biomimetic periosteum, which may have applications in bone repair.

Hypoxia-inducible factor 1α (HIF-1α) is a major determinant in the enhanced function of muscle-derived progenitors from MRL/MpJ mice.

Although the mouse strain Murphy Roths Large (MRL/MpJ) possesses high regenerative potential, the mechanism of tissue regeneration, including skeletal muscle, in MRL/MpJ mice after injury is still unclear. Our previous studies have shown that muscle-derived stem/progenitor cell (MDSPC) function is significantly enhanced in MRL/MpJ mice when compared with MDSPCs isolated from age-matched wild-type (WT) mice. Using mass spectrometry-based proteomic analysis, we identified increased expression of hypoxia-inducible factor (HIF) 1α target genes (expression of glycolytic factors and antioxidants) in sera from MRL/MpJ mice compared with WT mice. Therefore, we hypothesized that HIF-1α promotes the high muscle healing capacity of MRL/MpJ mice by increasing the potency of MDSPCs. We demonstrated that treating MRL/MpJ MDSPCs with dimethyloxalylglycine and CoCl2 increased the expression of HIF-1α and target genes, including angiogenic and cell survival genes. We also observed that HIF-1α activated the expression of paired box (Pax)7 through direct interaction with the Pax7 promoter. Furthermore, we also observed a higher myogenic potential of MDSPCs derived from prolyl hydroxylase (Phd) 3-knockout (Phd3-/-) mice, which displayed higher stability of HIF-1α. Taken together, our findings suggest that HIF-1α is a major determinant in the increased MDSPC function of MRL/MpJ mice through enhancement of cell survival, proliferation, and myogenic differentiation.-Sinha, K. M., Tseng, C., Guo, P., Lu, A., Pan, H., Gao, X., Andrews, R., Eltzschig, H., Huard, J. Hypoxia-inducible factor 1α (HIF-1α) is a major determinant in the enhanced function of muscle-derived progenitors from MRL/MpJ mice.

Induction of Transient Morula-Like Cells in Mice Through STAT3 Activation.

Developing in vitro cell models that faithfully replicate the molecular and functional traits of cells from the earliest stages of mammalian development presents a significant challenge. The strategic induction of signal transducer and activator of transcription 3 (STAT3) phosphorylation, coupled with carefully defined culture conditions, facilitates the efficient reprogramming of mouse pluripotent cells into a transient morula-like cell (MLC) state. The resulting MLCs closely mirror their in vivo counterparts, exhibiting not only molecular resemblance but also the ability to differentiate into both embryonic and extraembryonic lineages. This reprogramming approach provides valuable insights into controlled cellular fate choice and opens new opportunities for studying early developmental processes in a dish.

Chemical reprogramming for cell fate manipulation: Methods, applications, and perspectives.

Chemical reprogramming offers an unprecedented opportunity to control somatic cell fate and generate desired cell types including pluripotent stem cells for applications in biomedicine in a precise, flexible, and controllable manner. Recent success in the chemical reprogramming of human somatic cells by activating a regeneration-like program provides an alternative way of producing stem cells for clinical translation. Likewise, chemical manipulation enables the capture of multiple (stem) cell states, ranging from totipotency to the stabilization of somatic fates in vitro. Here, we review progress in using chemical approaches for cell fate manipulation in addition to future opportunities in this promising field.

Krüppel-like factor 5 rewires NANOG regulatory network to activate human naive pluripotency specific LTR7Ys and promote naive pluripotency.

Endogenous retroviruses (ERVs) have been reported to participate in pre-implantation development of mammalian embryos. In early human embryogenesis, different ERV sub-families are activated in a highly stage-specific manner. How the specificity of ERV activation is achieved remains largely unknown. Here, we demonstrate the mechanism of how LTR7Ys, the human morula-blastocyst-specific HERVH long terminal repeats, are activated by the naive pluripotency transcription network. We find that KLF5 interacts with and rewires NANOG to bind and regulate LTR7Ys; in contrast, the primed-specific LTR7s are preferentially bound by NANOG in the absence of KLF5. The specific activation of LTR7Ys by KLF5 and NANOG in pluripotent stem cells contributes to human-specific naive pluripotency regulation. KLF5-LTR7Y axis also promotes the expression of trophectoderm genes and contributes to the expanded cell potential toward extra-embryonic lineage. Our study suggests that HERVs are activated by cell-state-specific transcription machinery and promote stage-specific transcription network and cell potency.

From Vial to Vein: Crucial Gaps in Mesenchymal Stromal Cell Clinical Trial Reporting.

Retrospective analysis of clinical trial outcomes is a vital exercise to facilitate efficient translation of cellular therapies. These analyses are particularly important for mesenchymal stem/stromal cell (MSC) products. The exquisite responsiveness of MSCs, which makes them attractive candidates for immunotherapies, is a double-edged sword; MSC clinical trials result in inconsistent outcomes that may correlate with underlying patient biology or procedural differences at trial sites. Here we review 45 North American MSC clinical trial results published between 2015 and 2021 to assess whether these reports provide sufficient information for retrospective analysis. Trial reports routinely specify the MSC tissue source, autologous or allogeneic origin and administration route. However, most methodological aspects related to cell preparation and handling immediately prior to administration are under-reported. Clinical trial reports inconsistently provide information about cryopreservation media composition, delivery vehicle, post-thaw time and storage until administration, duration of infusion, and pre-administration viability or potency assessments. In addition, there appears to be significant variability in how cell products are formulated, handled or assessed between trials. The apparent gaps in reporting, combined with high process variability, are not sufficient for retrospective analyses that could potentially identify optimal cell preparation and handling protocols that correlate with successful intra- and inter-trial outcomes. The substantial preclinical data demonstrating that cell handling affects MSC potency highlights the need for more comprehensive clinical trial reporting of MSC conditions from expansion through delivery to support development of globally standardized protocols to efficiently advance MSCs as commercial products.

Evolution of Somite Compartmentalization: A View From Xenopus.

Somites are transitory metameric structures at the basis of the axial organization of vertebrate musculoskeletal system. During evolution, somites appear in the chordate phylum and compartmentalize mainly into the dermomyotome, the myotome, and the sclerotome in vertebrates. In this review, we summarized the existing literature about somite compartmentalization in Xenopus and compared it with other anamniote and amniote vertebrates. We also present and discuss a model that describes the evolutionary history of somite compartmentalization from ancestral chordates to amniote vertebrates. We propose that the ancestral organization of chordate somite, subdivided into a lateral compartment of multipotent somitic cells (MSCs) and a medial primitive myotome, evolves through two major transitions. From ancestral chordates to vertebrates, the cell potency of MSCs may have evolved and gave rise to all new vertebrate compartments, i.e., the dermomyome, its hypaxial region, and the sclerotome. From anamniote to amniote vertebrates, the lateral MSC territory may expand to the whole somite at the expense of primitive myotome and may probably facilitate sclerotome formation. We propose that successive modifications of the cell potency of some type of embryonic progenitors could be one of major processes of the vertebrate evolution.

Molecular differences of adipose-derived mesenchymal stem cells between non-responders and responders in treatment of transphincteric perianal fistulas.

BACKGROUND: Injection of autologous adipose tissue (AT) has recently been demonstrated to be an effective and safe treatment for anal fistulas. AT mesenchymal stem cells (AT-MSCs) mediate the healing process, but the relationship between molecular characteristics of AT-MSCs of the injected AT and fistula healing has not been adequately studied. Thus we aimed to characterize the molecular and functional properties of AT-MSCs isolated from autologous AT injected as a treatment of cryptogenic high transsphincteric perianal fistulas and correlate these findings to the healing process. METHODS: 27 patients (age 45 ± 2 years) diagnosed with perianal fistula were enrolled in the study and treated with autologous AT injected around the anal fistula tract. AT-MSCs were isolated for cellular and molecular analyses. The fistula healing was evaluated by MRI scanning after 6 months of treatment. AT-MSC phenotype was compared between responders and non-responders with respect to fistula healing. RESULTS: 52% of all patients exhibited clinical healing of the fistulas as evaluated 6 months after last injection. Cultured AT-MSCs in the responder group had a lower short-term proliferation rate and higher osteoblast differentiation potential compared to non-responder AT-MSCs. On the other hand, adipocyte differentiation potential of AT-MSCs was higher in non-responder group. Interestingly, AT-MSCs of responders exhibited lower expression of inflammatory and senescence associated genes such as IL1B, NFKB, CDKN2A, TPB3,TGFB1. CONCLUSION: Our data suggest that cellular quality of the injected AT-MSCs including cell proliferation, differentiation capacity and secretion of proinflammatory molecules may provide a possible mechanism underlying fistula healing. Furthermore, these biomarkers may be useful to predict a positive fistula healing outcome. TRIAL REGISTRATION: NTC04834609, Registered 6 April 2021. https://clinicaltrials.gov/ct2/show/NCT04834609.

The Zscan4-Tet2 Transcription Nexus Regulates Metabolic Rewiring and Enhances Proteostasis to Promote Reprogramming.

Evolutionarily conserved SCAN (named after SRE-ZBP, CTfin51, AW-1, and Number 18 cDNA)-domain-containing zinc finger transcription factors (ZSCAN) have been found in both mouse and human genomes. Zscan4 is transiently expressed during zygotic genome activation (ZGA) in preimplantation embryos and induced pluripotent stem cell (iPSC) reprogramming. However, little is known about the mechanism of Zscan4 underlying these processes of cell fate control. Here, we show that Zscan4f, a representative of ZSCAN proteins, is able to recruit Tet2 through its SCAN domain. The Zscan4f-Tet2 interaction promotes DNA demethylation and regulates the expression of target genes, particularly those encoding glycolytic enzymes and proteasome subunits. Zscan4f regulates metabolic rewiring, enhances proteasome function, and ultimately promotes iPSC generation. These results identify Zscan4f as an important partner of Tet2 in regulating target genes and promoting iPSC generation and suggest a possible and common mechanism shared by SCAN family transcription factors to recruit ten-eleven translocation (TET) DNA dioxygenases to regulate diverse cellular processes, including reprogramming.

Efficacy and safety of autologous cell therapies for knee cartilage defects (autologous stem cells, chondrocytes or the two): randomized controlled trial design.

AIM: The main aim of this trial is to test the safety and efficacy of autologous stromal/stem cells, chondrocytes or the two combined in the treatment of knee cartilage defects. PATIENTS & METHODS: Patients with symptomatic chondral/osteochondral defects will be randomized to cell therapy treatment with one of three cell populations (1:1:1). The primary efficacy outcome is a functional knee score (Lysholm) at 15 months post-treatment and the primary safety outcome is the incidence of adverse events. Secondary objectives are to analyze repair tissues, quality of life and cost-utility assessments. Exploratory objectives are to identify predictors for success/potency and dose-response relationships. RESULTS & CONCLUSION: This trial has been carefully designed so that valuable scientific and clinical information can be gathered throughout and in the final analysis.

Are surface antigens suited to verify the redifferentiation potential and culture purity of human chondrocytes in cell-based implants.

Cell expansion in vitro is a prequisite to obtain a sufficient quantity of cells for cell-based cartilage repair of articular cartilage lesions. During this process verification of redifferentiation potential of highly expanded chondrocytes is required. Furthermore, cellular impurities of chondrocyte cultures have to be excluded. For this purpose, redifferentiation of expanded human chondrocytes in passage 3 or 5 was initiated in bioresorbable polyglycolic acid-fibrin (PGA-fibrin) scaffolds and selected potential markers were analysed during the process of cell expansion and redifferentiation. Chondrocyte expansion was accompanied by a decrease of collagen type II and COMP and an increase of collagen type I expression indicating cell dedifferentiation. Redifferentiation of chondrocytes in PGA-fibrin scaffolds was accompanied by an increase of collagen II/I ratio. Flow cytometric analyses revealed that in contrast to CD44 and CD49e, CD63 and CD166 showed significant changes in the number of positive cells during redifferentiation. CD14 and CD45 are not expressed by chondrocytes and are therefore possible candidates to detect specifically monocytes or haematopoetic cells in chondrocyte cultures. Characterization of surface antigen expression revealed two promising candidates (CD63 and CD166) to describe the process of redifferentiation, while CD14 and CD45 are suitable markers to exclude impurities by monocytes or haematopoetic cells.

Unrestricted lineage differentiation of parthenogenetic ES cells.

The developmental potential of parthenogenetic embryonic stem (P-ES) cells was studied in teratomas and mouse chimaeras. Teratomas derived from P-ES cells contained a mixture of tissue types with variable proportions of specific tissues. Three of the eight P-ES cell lines analysed showed high proportions of striated muscle in teratomas, similar to teratomas from normal embryos or ES cell lines derived from fertilised embryos (F-ES cells). Our study also revealed that one P-ES cell line showed little lineage restriction in injection chimaeras. Descendants of the P-ES cells contributed to most tissues of chimaeric fetuses in patterns similar to F-ES cells. Normal colonisation of muscle, liver and pancreas was found in adult chimaeras. P-ES cells also showed similar haematopoietic differentiation and maturation as F-ES cells. However, extensive P-ES cell contribution was associated with a reduction in body size. These findings suggest that, while P-ES cells display more extensive developmental potential than the cells of parthenogenetic embryos from which they were derived, they only retained properties related to the presence of the maternal genome. To elucidate the molecular basis for the lack of lineage restriction during in vivo differentiation, the expression of four imprinted genes, H19, Igf2r, Igf2 and Snrpn was compared among five P-ES and two F-ES cell lines. Expression levels of these genes varied among the different ES cell lines, both in undifferentiated ES cells and in embryoid bodies.

Discovery of a Potent, Injectable Inhibitor of Aurora Kinases Based on the Imidazo-[1,2-a]-Pyrazine Core.

The imidazo-[1,2-a]-pyrazine (1) is a dual inhibitor of Aurora kinases A and B with modest cell potency (IC50 = 250 nM) and low solubility (5 µM). Lead optimization guided by the binding mode led to the acyclic amino alcohol 12k (SCH 1473759), which is a picomolar inhibitor of Aurora kinases (TdF K d Aur A = 0.02 nM and Aur B = 0.03 nM) with improved cell potency (phos-HH3 inhibition IC50 = 25 nM) and intrinsic aqueous solubility (11.4 mM). It also demonstrated efficacy and target engagement in human tumor xenograft mouse models.