Constructing human neural circuits in living systems by transplantation.

Our understanding of how the brain assembles its circuits and how this goes awry in disease remains incomplete. There has been great progress in generating human neurons from stem cells in vitro and, more recently, in constructing circuits with human cells in vivo by transplantation. Here, I highlight approaches, promises, and challenges of growing human neurons in living animals to study human development and disease.

Developmental and stem cell biology's bright future.

The next 50 years of developmental biology will illuminate exciting new discoveries but are also poised to provide solutions to important problems society faces. Ten scientists whose work intersects with developmental biology in various capacities tell us about their vision for the future.

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.

Redefining intestinal stemness: The emergence of a new ISC population.

In tissue homeostasis, intestinal stem cells (ISCs) undergo continuous self-renewal to sustain rapid cellular turnover. In this issue of Cell, Capdevila et al.1 and Malagola, Vasciaveo, et al.2 identify a new ISC population in the upper crypt that can generate Lgr5+ stem cells during homeostasis.

Tracing the origin of alveolar stem cells in lung repair and regeneration.

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

Dynamic regulation of tissue fluidity controls skin repair during wound healing.

During wound healing, different pools of stem cells (SCs) contribute to skin repair. However, how SCs become activated and drive the tissue remodeling essential for skin repair is still poorly understood. Here, by developing a mouse model allowing lineage tracing and basal cell lineage ablation, we monitor SC fate and tissue dynamics during regeneration using confocal and intravital imaging. Analysis of basal cell rearrangements shows dynamic transitions from a solid-like homeostatic state to a fluid-like state allowing tissue remodeling during repair, as predicted by a minimal mathematical modeling of the spatiotemporal dynamics and fate behavior of basal cells. The basal cell layer progressively returns to a solid-like state with re-epithelialization. Bulk, single-cell RNA, and epigenetic profiling of SCs, together with functional experiments, uncover a common regenerative state regulated by the EGFR/AP1 axis activated during tissue fluidization that is essential for skin SC activation and tissue repair.

Time-resolved fate mapping identifies the intestinal upper crypt zone as an origin of Lgr5+ crypt base columnar cells.

In the prevailing model, Lgr5+ cells are the only intestinal stem cells (ISCs) that sustain homeostatic epithelial regeneration by upward migration of progeny through elusive upper crypt transit-amplifying (TA) intermediates. Here, we identify a proliferative upper crypt population marked by Fgfbp1, in the location of putative TA cells, that is transcriptionally distinct from Lgr5+ cells. Using a kinetic reporter for time-resolved fate mapping and Fgfbp1-CreERT2 lineage tracing, we establish that Fgfbp1+ cells are multi-potent and give rise to Lgr5+ cells, consistent with their ISC function. Fgfbp1+ cells also sustain epithelial regeneration following Lgr5+ cell depletion. We demonstrate that FGFBP1, produced by the upper crypt cells, is an essential factor for crypt proliferation and epithelial homeostasis. Our findings support a model in which tissue regeneration originates from upper crypt Fgfbp1+ cells that generate progeny propagating bi-directionally along the crypt-villus axis and serve as a source of Lgr5+ cells in the crypt base.

Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

Early rhombic lip Protogenin+ve stem cells in a human-specific neurovascular niche initiate and maintain group 3 medulloblastoma.

We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.

Spin glasses, error correcting codes, and synchronization of human stem cell organoids.

Magnetic spins, pendulum clocks, and fireflies all self-organize into coherent collectives when arranged into groups of spatially coupled and interacting individuals. Ramanathan and colleagues demonstrate that spatial coupling of human stem cell organoids induces coherent progression through developmental transitions, allowing the dissection of molecular circuits underlying human development.

Comparative landscape of genetic dependencies in human and chimpanzee stem cells.

Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether human cells exhibit distinct genetic dependencies. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell-cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells and cerebral organoids, supporting the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells reshaped the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.

Trellis tree-based analysis reveals stromal regulation of patient-derived organoid drug responses.

Patient-derived organoids (PDOs) can model personalized therapy responses; however, current screening technologies cannot reveal drug response mechanisms or how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly multiplexed mass cytometry platform to measure post-translational modification (PTM) signaling, DNA damage, cell-cycle activity, and apoptosis in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment-specific drug responses in thousands of single-cell datasets, we developed "Trellis"-a highly scalable, tree-based treatment effect analysis method. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is rarer, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate PDO plasticity-shifting proliferative colonic stem cells (proCSCs) to slow-cycling revival colonic stem cells (revCSCs) to protect cancer cells from chemotherapy.

Seven days in the life cycle of Homo sapiens.

The second week of embryonic development is a critical phase of the human life cycle and one that has been largely inaccessible to scientific investigation. Recent studies of human embryo models built from stem cells promise to yield dramatic insights into the key events of cell specification and morphogenesis that occur during this brief window of embryogenesis.

Targeted alveolar regeneration with Frizzled-specific agonists.

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.

Hallmarks of aging: An expanding universe.

Aging is driven by hallmarks fulfilling the following three premises: (1) their age-associated manifestation, (2) the acceleration of aging by experimentally accentuating them, and (3) the opportunity to decelerate, stop, or reverse aging by therapeutic interventions on them. We propose the following twelve hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. These hallmarks are interconnected among each other, as well as to the recently proposed hallmarks of health, which include organizational features of spatial compartmentalization, maintenance of homeostasis, and adequate responses to stress.

Tracking cell-type-specific temporal dynamics in human and mouse brains.

Progenitor cells are critical in preserving organismal homeostasis, yet their diversity and dynamics in the aged brain remain underexplored. We introduced TrackerSci, a single-cell genomic method that combines newborn cell labeling and combinatorial indexing to characterize the transcriptome and chromatin landscape of proliferating progenitor cells in vivo. Using TrackerSci, we investigated the dynamics of newborn cells in mouse brains across various ages and in a mouse model of Alzheimer's disease. Our dataset revealed diverse progenitor cell types in the brain and their epigenetic signatures. We further quantified aging-associated shifts in cell-type-specific proliferation and differentiation and deciphered the associated molecular programs. Extending our study to the progenitor cells in the aged human brain, we identified conserved genetic signatures across species and pinpointed region-specific cellular dynamics, such as the reduced oligodendrogenesis in the cerebellum. We anticipate that TrackerSci will be broadly applicable to unveil cell-type-specific temporal dynamics in diverse systems.

An oncogenic phenoscape of colonic stem cell polarization.

Cancer cells are regulated by oncogenic mutations and microenvironmental signals, yet these processes are often studied separately. To functionally map how cell-intrinsic and cell-extrinsic cues co-regulate cell fate, we performed a systematic single-cell analysis of 1,107 colonic organoid cultures regulated by (1) colorectal cancer (CRC) oncogenic mutations, (2) microenvironmental fibroblasts and macrophages, (3) stromal ligands, and (4) signaling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation phenoscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSCs) to oncogene-driven LRIG1+ hyper-proliferative CSCs (proCSCs). The transition from revCSCs to proCSCs is regulated by decreasing WNT3A and TGF-ß-driven YAP signaling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find that APC loss and KRASG12D collaboratively limit access to revCSCs and disrupt stromal-epithelial communication-trapping epithelia in the proCSC fate. These results reveal that oncogenic mutations dominate homeostatic differentiation by obstructing cell-extrinsic regulation of cell-fate plasticity.

Cerebrovasculature pumps up progenitors.

Fetal human brain stem cell niches that contain multipotent neural progenitors are progressively vascularized during development. Crouch et al. (Crouch et al., 2022) report endothelial and mural lineage trajectories that build developing prenatal vascular in second trimester fetal brain. This cerebral angiogenesis in neural progenitor zones occurs simultaneously with and can promote neurogenesis.

Studying human neural function in vivo at the cellular level: Chasing chimeras?

Despite its importance to understanding human brain (dys)function, it has remained challenging to study human neurons in vivo. Recent approaches, using transplantation of human cortical neurons into the rodent brain, offer new prospects for the study of human neural function and disease in vivo, from molecular to circuit levels.