Expansion Of Progenitor Cells Research Articles

8649 Ablation of Leptin Receptors in Somatotropes Impacts Transcriptomic Plasticity in the Pou1f1 Lineage of Female Pituitary Cells

Abstract Disclosure: T.K. Miles: None. A.K. Odle: None. S. Byrum: None. A. Lagasse: None. A. Haney: None. V.G. Ortega: None. A. Herdman: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. Anterior pituitary somatotropes produce growth hormone (GH) to optimize body composition, receiving tropic signals from leptin about the metabolic state. When leptin receptors are ablated in somatotropes, GH proteins, serum GH and GHRH receptors are reduced. Unexpectedly, POU1F1, Prolactin, and TSH-beta proteins are also reduced in pituitaries of Somatotrope LEPR-null females (mutants), suggesting a tropic role for leptin in differentiation. To test this hypothesis, single cell RNA-seq analysis compared the pituitary transcriptome of 6-month-old control and mutant female mice housed under thermoneutral conditions (28°C). Following scRNA-seq, cells were clustered computationally with the use of Seurat; all cell types were identified in separate UMAP clusters. Mutants showed increases in cell numbers in somatotrope (4.7X) and Pou1f1 (5.7x) clusters, but no change in thyrotrope or lactotrope clusters. In LEPR-null female somatotropes, the 516 differentially expressed genes (DEGs; adj p<0.05) included upregulated Pomc, Prl, Lhb, Tshb and Cga (Log2FC+0.5-1.7) and downregulated Pou1f1, Ghrhr, and Gh (Log2FC -0.5—1.5). In the lactotrope cluster from mutants, Pomc, Cga, and Gh were upregulated (Log2FC+1.1-1.5) and Pou1f1 and Prl were downregulated. The thyrotrope cluster was unchanged. Prl, Pomc, and Gh were upregulated in the Pou1f1 cluster. The down regulation of Gh, Pou1f1, and Ghrhr in somatotropes and Prl in lactotropes correlates with the lower serum levels of GH and Prl in mutant females. However, the upregulated DEGs do not correlate with increased secretory activity suggesting that the multihormonal cells are not fully functional. This was confirmed with Ingenuity Pathway Analysis (IPA) showing downregulation of canonical pathways involved in secretion in mutant somatotropes. Unexpectedly, mutant females exposed to a HFD (60% fat) for 16 weeks (+16.6g) showed a reversal of the changes in somatotrope cell numbers and multihormonal expression seen in control fed mutants. The increase in genes belonging to IPA canonical cytoplasmic translation pathways in mutant somatotropes were also completely reversed following a HFD. Furthermore, the downregulated secretory/signaling pathways seen in control fed mutant female somatotropes and lactotropes (endocytosis, vesicle transport, Gaq, GnRH) were upregulated modestly following a HFD. The high serum leptin in the HFD fed mutant mice may activate signaling pathways in cells still expressing LEPR. Collectively, these results confirm a role for leptin in maintaining differentiated somatotropes. They also suggest that leptin may limit expansion of multihormonal progenitor cells. The decreased multihormonal expression and protein synthesis in lactotrope clusters from mutant mice exposed to a HFD are also seen in lactotropes from control mice on a HFD, which suggests that a HFD may compromise pituitary plasticity. Presentation: 6/3/2024

A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

Generation of human expandable limb-bud-like progenitors via chemically induced dedifferentiation.

In certain highly regenerative animals, cellular dedifferentiation occurs after injury, allowing specialized cells to become progenitor cells for regeneration. However, this capacity is restricted in human cells due to reduced plasticity. Here, we introduce a chemical-induced dedifferentiation approach that reverts the differentiated cells to a progenitor-like state, conferring the features of human limb bud cells from human adult somatic cells. These chemically induced human limb-bud-like progenitors (hCiLBP cells) show a high degree of transcriptomic similarity to human embryonic limb bud progenitors. Importantly, we established culture conditions that allow hCiLBP cells to undergo extensive expansion while maintaining population homogeneity and long-term self-renewal capacity. Moreover, hCiLBP cells exhibit increased osteochondrogenic differentiation ability, providing an innovative platform for generation of skeletal lineage cell types. These results highlight a potential therapeutic approach for repairing damaged human tissues through reversal of developmental pathways from mature cells to expandable progenitor cells.

P53 mutation biases squamocolumnar junction progenitor cells towards dysplasia rather than metaplasia in Barrett’s oesophagus

BackgroundWhile p53 mutations occur early in Barrett’s oesophagus (BE) progression to oesophageal adenocarcinoma (EAC), their role in gastric cardia stem cells remains unclear.ObjectiveThis study investigates the impact of p53 mutation...

Further biological characterization of small molecules UM171 and SR1: In vitro effects on three hematopoietic cell populations from human cord blood

Small molecules UM171 and SR1 have already been taken into clinically-oriented protocols for the ex vivo expansion of hematopoietic stem (HSCs) and progenitor (HPCs) cells. In order to gain further insight into their biology, in the present study we have assessed their effects, both individually and in combination, on the in vitro long-term proliferation and expansion of HSCs and HPCs contained within three different cord blood-derived cell populations: MNCs (CD34+ cells = 0.8 %), LIN− cells (CD34+ cells = 41 %), and CD34+ cells (CD34+ cells >98 %). Our results show that when added to cultures in the absence of recombinant stimulatory cytokines, neither molecule had any effect. In contrast, when added in the presence of hematopoietic cytokines, UM171 and SR1 had significant stimulatory effects on cell proliferation and expansion in cultures of LIN− and CD34+ cells. No significant effects were observed in cultures of MNCs. The effects of both molecules were more pronounced in cultures with the highest proportion of CD34+ cells, and the greatest effects were observed when both molecules were added in combination. In the absence of small molecules, cell numbers reached a peak by days 25–30, and then declined; whereas in the presence of UM171 or/and SR1 cell numbers were sustained up to day 45 of culture. Our results indicate that besides CD34+ cells, LIN− cells could also be used as input cells in clinically-oriented expansion protocols, and that using both molecules simultaneously would be a better approach than using only one of them.

MTORC1 mediates the expansion of hematopoietic stem and progenitor cells through ribosome biogenesis protein Urb2 in zebrafish

Mammalian target of rapamycin (mTOR) serves as the key sensor to control protein synthesis, cell growth, and survival. Despite mTOR is reported to regulate hematopoietic stem and progenitor cell (HSPC) engraftment and multiple-lineage hematopoiesis in mice, the roles of unique mTOR complexes (mTORCs) in early HSPC development and HSPC pool formation have not been adequately elucidated. Here, we uncover that mTORC1 is essential for early HSPC expansion in zebrafish. mTORC1 signaling was highly activated in definitive HSPCs during the emerging and expanding stages. Pharmacological or genetic inactivation of mTORC1 would cause defective HSPC expansion and migration due to disrupted cell proliferation. Interestingly, mTORC2 is dispensable for early HSPC development. Ribosome biogenesis protein Urb2 was downregulated upon mTORC1 inhibition, and urb2 overexpression partially rescued the hematopoietic defects in mTORC1-deficient embryos. These data demonstrate that mTORC1 signaling regulates early HSPC expansion through Urb2, and this work will deepen our understanding of mTOR in different physiological processes.

CHIP: a clonal odyssey of the bone marrow niche.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying somatic mutations. While CHIP is typically asymptomatic, it has garnered substantial attention due to its association with the pathogenesis of multiple disease conditions, including cardiovascular disease (CVD) and hematological malignancies. In this Review, we will discuss seminal and recent studies that have advanced our understanding of mechanisms that drive selection for mutant HSPCs in the BM niche. Next, we will address recent studies evaluating potential relationships between the clonal dynamics of CHIP and hematopoietic development across the lifespan. Next, we will examine the roles of systemic factors that can influence hematopoietic stem cell (HSC) fitness, including inflammation, and exposures to cytotoxic agents in driving selection for CHIP clones. Furthermore, we will consider how - through their impact on the BM niche - lifestyle factors, including diet, exercise, and psychosocial stressors, might contribute to the process of somatic evolution in the BM that culminates in CHIP. Finally, we will review the role of old age as a major driver of selection in CHIP.

Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages.

Conventional treatments exhibit various side effects on chronic stress anemia. Extramedullary stress erythropoiesis is a compensatory mechanism, which may effectively counteract anemia. Angelica sinensis polysaccharides (ASP) are the main active ingredient found in Angelica sinensis and exhibit antioxidant and hematopoietic effects. However, the effects of ASP on extramedullary stress erythropoiesis remain to be unclear. Here, we demonstrated the protective effects of ASP on chemotherapeutic drug 5-fluorouracil (5-FU)-induced decline in peripheral blood parameters such as RBC counts, HGB, HCT, and MCH, and the decline of BFU-E colony enumeration in the bone marrow. Meanwhile, ASP promoted extramedullary erythropoiesis, increasing cellular proliferation in the splenic red pulp and cyclin D1 protein expression, abrogating phase G0/G1 arrest of c-kit+ cells in mouse spleen. RT-qPCR and immunohistochemistry further revealed that ASP increased macrophage chemokine Ccl2 genetic expression and the number of F4/80+ macrophages in the spleen. The colony-forming assay showed that ASP significantly increased splenic BFU-E. Furthermore, we found that ASP facilitated glycolytic genes including Hk2, Pgk1, Pkm, Pdk1, and Ldha via PI3K/Akt/HIF2α signaling in the spleen. Subsequently, ASP declined pro-proinflammatory factor IL-1β, whereas upregulating erythroid proliferation-associated genes Gdf15, Bmp4, Wnt2b, and Wnt8a. Moreover, ASP facilitated EPO/STAT5 signaling in splenic macrophages, thus enhancing erythroid lineage Gata2 genetic expression. Our study indicated that ASP may improve glycolysis, promoting the activity of splenic macrophages, subsequently promoting erythroid progenitor cell expansion. Additionally, ASP facilitates erythroid differentiation via macrophage-mediated EpoR/STAT5 signaling; suggesting it might be a promising strategy for stress anemia treatment.

Expansion of human allogeneic liver-derived progenitor cells for liver regenerative therapy in serum-free culture conditions

Human allogeneic liver-derived progenitor cells (HALPCs) display advanced ability to differentiate into hepatocyte-like cells and exhibit potent immunomodulatory, anti-inflammatory, and anti-fibrotic properties. HALPCs have been successfully manufactured under good manufacturing practice (GMP) and are currently in clinical development. A previous phase 2a trial demonstrated the safety of peripheral intravenous infusions of HALPCs and preliminary evidence of the cells’ properties to restore liver function in patients with acute-on-chronic liver failure (ACLF), thus potentially improving their survival. A phase 2b trial is currently ongoing across multiple centers (NCT04229901) to obtain proof-of-concept on efficacy and additional safety. HALPCs are currently manufactured using fetal bovine serum (FBS), which can reveal qualitative and quantitative variations between batches. The use of serum-free medium (SFM) represents an alternative means to overcome this variability while also complying fully with regulations. The aim of this study was to compare current FBS-containing culture conditions with two industry-available GMP-compliant SFMs: StemMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and PRIME-XV (FUJIFILM Irvine Scientific, Santa Ana, California, USA).The proliferation of HALPCs was significantly stimulated by both SFMs, which shortened both their emergence period and population doubling time. This effect was correlated with a significant improvement in their genetic stability as analyzed by conventional karyotyping. The expression profile (identity and purity) and functionality of HALPCs cultured in SFM were maintained, as demonstrated by flow cytometry and enzyme-linked immunoassay (ELISA), respectively. Their potency, evaluated via prostaglandin E2 (PGE2) secretion, showed a similar effect on CD4+ T-cell proliferation in FBS and SFM conditions. Furthermore, a greater proportion of HALPCs cultured in SFM showed enhanced expression of tissue factor (CD142) compared with the FBS condition.Altogether, SFM conditions enabled consistent HALPC quality to be achieved without altering their expression and functional profiles.

Oncogenic Calreticulin Induces Immune Escape by Stimulating TGFβ Expression and Regulatory T-cell Expansion in the Bone Marrow Microenvironment.

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFβ1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFβ antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFβ. TGFβ1 reduced perforin and TNFα production by T cells in vitro. TGFβ1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFβ1 expression. In four independent patient cohorts, TGFβ1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFβ1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFβ1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFβ antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms.

Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

BackgroundThe aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell–matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions. As over-expression of the laminin-binding α6 integrin (Itgα6) is associated with poor prognosis and reduced survival in breast cancer, we here investigate the role of Itgα6 in mammary tumorigenesis.MethodsWe used Blg-Cre; Brca1F/F; Trp53F/F mice, a model that phenocopies human basal-like breast cancer with BRCA1 mutations. We generated mutant mice proficient or deficient in Itgα6 expression and followed tumor formation. Mammary tumors and pretumoral tissues were characterized by immunohistochemistry, flow cytometry, RT-qPCR, Western blotting and organoid cultures. Clonogenicity of luminal progenitors from preneoplastic glands was studied in 3D Matrigel cultures.ResultsWe show that Itga6 deletion favors activation of p16 cell cycle inhibitor in the preneoplastic tissue. Subsequently, the amplification of luminal progenitors, the cell of origin of Brca1-deficient tumors, is restrained in Itgα6-deficient gland. In addition, the partial EMT program operating in Brca1/p53-deficient epithelium is attenuated in the absence of Itgα6. As a consequence of these events, mammary tumor formation is delayed in Itgα6-deficient mice. After tumor formation, the lack of Itgα6 does not affect tumor growth but rather alters their differentiation, resulting in reduced expression of basal cell markers.ConclusionsOur data indicate that Itgα6 has a pro-tumorigenic role in Blg-Cre; Brca1F/F; Trp53F/F mice developing basal-like mammary tumors. In particular, we reveal that Itgα6 is required for the luminal progenitor expansion and the aberrant partial EMT program that precedes the formation of BRCA1 deficient tumors.

Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Jag1/2 maintain esophageal homeostasis and suppress foregut tumorigenesis by restricting the basal progenitor cell pool

Basal progenitor cells are crucial for maintaining foregut (the esophagus and forestomach) homeostasis. When their function is dysregulated, it can promote inflammation and tumorigenesis. However, the mechanisms underlying these processes remain largely unclear. Here, we employ genetic mouse models to reveal that Jag1/2 regulate esophageal homeostasis and foregut tumorigenesis by modulating the function of basal progenitor cells. Deletion of Jag1/2 in mice disrupts esophageal and forestomach epithelial homeostasis. Mechanistically, Jag1/2 deficiency impairs activation of Notch signaling, leading to reduced squamous epithelial differentiation and expansion of basal progenitor cells. Moreover, Jag1/2 deficiency exacerbates the deoxycholic acid (DCA)-induced squamous epithelial injury and accelerates the initiation of squamous cell carcinoma (SCC) in the forestomach. Importantly, expression levels of JAG1/2 are lower in the early stages of human esophageal squamous cell carcinoma (ESCC) carcinogenesis. Collectively, our study demonstrates that Jag1/2 are important for maintaining esophageal and forestomach homeostasis and the onset of foregut SCC.

In vitro and in vivo evaluation of periosteum-derived cells and iPSC-derived chondrocytes encapsulated in GelMA for osteochondral tissue engineering.

Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.

TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Lentivirus-Mediated BCL-XL Overexpression Inhibits Stem Cell Apoptosis during Ex Vivo Expansion and Provides Competitive Advantage Following Xenotransplantation.

Hematopoietic reconstitution after hematopoietic stem cell transplantation (HSCT) is influenced by the number of transplanted cells. However, under certain conditions donor cell counts are limited and impair clinical outcome. Hematopoietic stem and progenitor cell (HSPC) expansion prior to HSCT is a widely used method to achieve higher donor cell counts and minimize transplantation-related risks such as graft failure or delayed engraftment. Still, expansion in a non-physiological environment can trigger cell death mechanisms and hence counteract the desired effect. We have shown earlier that during HSCT a relevant amount of HSPCs were lost due to apoptosis and that cell death inhibition in donor HSPCs improved engraftment in xenotransplantation experiments. Here, we assessed the effect of combined ex vivo expansion and cell death inhibition on HSPC yield and their reconstitution potential in vivo. During expansion with cytokines and the small molecule inhibitor StemRegenin 1, concomitant lentiviral overexpression of antiapoptotic BCL-XL resulted in an increased yield of transduced HSPCs. Importantly, BCL-XL overexpression enhanced the reconstitution potential of HSPCs in xenotransplantation experiments in vivo. In contrast, treatment with caspase and necroptosis inhibitors had no favorable effects on HSPC yields nor on cell viability. We postulate that overexpression of antiapoptotic BCL-XL, both during ex vivo expansion and transplantation, is a promising approach to improve the outcome of HSCT in situations with limited donor cell numbers. However, such apoptosis inhibition needs to be transient to avoid long-term sequelae like leukemia.

Hematopoiesis after anti-CD117 monoclonal antibody treatment in the settings of wild-type and Fanconi anemia mice.

Anti-CD117 monoclonal antibody (mAb) agents have emerged as exciting alternative conditioning strategies to traditional genotoxic irradiation or chemotherapy for both allogeneic and autologous gene-modified hematopoietic stem cell transplantation. Furthermore, these agents are concurrently being explored in the treatment of mast cell disorders. Despite promising results in animal models and more recently in patients, the short- and long-term effects of these treatments have not been fully explored. We conducted rigorous assessments to evaluate the effects of an antagonistic anti-mCD117 mAb, ACK2, on hematopoiesis in wild-type and Fanconi anemia (FA) mice. Importantly, we found no evidence of short-term DNA damage in either setting following this treatment, suggesting that ACK2 does not induce immediate genotoxicity, providing crucial insights into its safety profile. Surprisingly, FA mice exhibited an increase in colony formation after ACK2 treatment, indicating a potential targeting of hematopoietic stem cells and expansion of hematopoietic progenitor cells. Moreover, the long-term phenotypic and functional changes in hematopoietic stem and progenitor cells did not differ significantly between the ACK2-treated and control groups, in either setting, suggesting that ACK2 does not adversely affect hematopoietic capacity. These findings underscore the safety of these agents when utilized as a short-course treatment in the context of conditioning, as they did not induce significant DNA damage in hematopoietic stem or progenitor cells. However, single-cell RNA sequencing, used to compare gene expression between untreated and treated mice, revealed that the ACK2 mAb, via c-Kit downregulation, effectively modulated the MAPK pathway with Fos downregulation in wild-type and FA mice. Importantly, this modulation was achieved without causing prolonged disruptions. These findings validate the safety of anti-CD117 mAb treatment and also enhance our understanding of its intricate mode of action at the molecular level.

In vivo and in vitro effects of cord blood hematopoietic stem and progenitor cell (HSPC) expansion using valproic acid and/or nicotinamide

BackgroundHigh self-renewal capacity and most permissive nature of umbilical cord blood (CB) results with successful transplant outcomes but low hematopoietic stem and progenitor cell (HSPC) counts limits wider use. In order to overcome this problem ex vivo expansion with small molecules such as Valproic acid (VPA) or Nicotinamide (NAM) have been shown to be effective. To the best of our knowledge, the combinatory effects of VPA and NAM on HSPC expansion has not been studied earlier. The aim of this study was to analyze ex vivo and in vivo efficacy of VPA and NAM either alone or in combination in terms of expansion and engraftment. MethodsA total of 44 CB units were included in this study. To determine the ex vivo and in vivo efficacy, human CB CD34+ cells were expanded with VPA and/or NAM and colony forming unit (CFU) assay was performed on expanded HSPC. Xenotransplantation was performed simultaneously by intravenous injection of expanded HSPC to NOD-SCID gamma (NSG) mice (n = 22). Significance of the difference between the expansion groups or xenotransplantation models was analyzed using t-test, Mann-Whitney, ANOVA or Kruskal-Wallis tests as appropriate considering the normality of distributions and the number of groups analyzed. ResultsIn vitro CD34+ HSPC expansion fold relative to cytokines-only was significantly higher with VPA compared to NAM [2.23 (1.07–5.59) vs 1.48 (1.00–4.40); p < 0.05]. Synergistic effect of VPA+NAM has achieved a maximum relative expansion fold at 21 days (D21) of incubation [2.95 (1.00–11.94)]. There was no significant difference between VPA and VPA+NAM D21 (p = 0.44). Fold number of colony-forming unit granulocyte-macrophage (CFU-GM) colonies relative to the cytokine-only group was in favor of NAM compared to VPA [1.87 (1.00–3.59) vs 1.00 (1.00–1.81); p < 0.01]. VPA+NAM D21 [1.62 (1.00–2.77)] was also superior against VPA (p < 0.05). There was no significant difference between NAM and VPA+NAM D21. Following human CB34+ CB transplantation (CBT) in the mouse model, fastest in vivo leukocyte recovery was observed with VPA+NAM expanded cells (6 ± 2 days) and the highest levels of human CD45 chimerism was detectable with VPA-expanded CBT (VPA: 5.42 % at day 28; NAM: 2.45 % at day 31; VPA+NAM 1.8 % at day 31). ConclusionOur study results suggest using VPA alone, rather than in combination with NAM or NAM alone, to achieve better and faster expansion and engraftment of CB HSPC.

The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis

Pre-mRNA splicing is a precise regulated process, and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs), and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remain largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout zebrafish line, and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells (RPCs) by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 knockout zebrafish line, we discovered that prpf31 knockout zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31 deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.

Mitigation of Osteoclast-Mediated Arthritic Bone Remodeling By Short Chain Fatty Acids.

The objective for this study was to evaluate the effects of short chain fatty acids (SCFAs) on arthritic bone remodeling. We treated a recently described preclinical murine model of psoriatic arthritis (PsA), R26STAT3Cstopfl/fl CD4Cre mice, with SCFA-supplemented water. We also performed in vitro osteoclast differentiation assays in the presence of serum-level SCFAs to evaluate the direct impact of these microbial metabolites on maturation and function of osteoclasts. We further characterized the molecular mechanism of SCFAs by transcriptional analysis. The osteoporosis condition in R26STAT3Cstopfl/fl CD4Cre animals is attributed primarily to robust osteoclast differentiation driven by an expansion of osteoclast progenitor cells (OCPs), accompanied by impaired osteoblast development. We show that SCFA supplementation can rescue the osteoporosis phenotype in this model of PsA. Our in vitro experiments revealed an inhibitory effect of the SCFAs on osteoclast differentiation, even at very low serum concentrations. This suppression of osteoclast differentiation enabled SCFAs to impede osteoporosis development in R26STAT3Cstopfl/fl CD4Cre mice. Further interrogation revealed that bone marrow-derived OCPs from diseased mice expressed a higher level of SCFA receptors than those of control mice and that the progenitor cells in the bone marrow of SCFA-treated mice presented a modified transcriptomic landscape, suggesting a direct impact of SCFAs on bone marrow progenitors in the context of osteoporosis. We demonstrated how gut microbiota-derived SCFAs can regulate distal pathology (ie, osteoporosis) and identified a potential therapeutic option for restoring bone density in rheumatic disease, further highlighting the critical role of the gut-bone axis in these disorders.