Platelets are produced via a canonical pathway emanating from multilineage hematopoietic stem cells (ML-HSCs) and a faster, direct pathway used by platelet-biased (P-) HSCs where the latter circumvents progenitors intermediate between HSCs and megakaryocytes. However, the physiological role of these distinct pathways remains to be determined. Here, we use Vldlr-iCre lineage tracing to identify platelets originating from platelet-biased HSCs and to show that P-HSC contribution to the megakaryocyte progenitor (MkP) population and platelet production is increased after 5-fluorouracil or cyclophosphamide treatment, where proliferating progenitors are severely depleted. In contrast, antibody-mediated platelet depletion, which leaves the progenitor hierarchy intact, did not increase the proportion of platelets generated by P-HSCs. Therefore, P-HSCs provide rapid platelet reconstitution without the need for complete re-establishment of the progenitor hierarchy after cytotoxic and genotoxic challenges due to their ability to more rapidly generate megakaryocyte progenitors from HSCs.

Platelet-biased hematopoietic stem cells (PLT-HSCs) play key roles in normal physiology, aging, and blood cancer. However, currently, no markers allow their accurate identification or prospective isolation. We here combine single-mouse hematopoietic stem cell (HSC) gene expression, chromatin accessibility, and surface proteome profiling to identify subtype-specific markers. Using machine learning, we identified markers (CD61hiCD274hiCD357loCD27lo) that isolate PLT-HSCs to high purity, validated by single-cell transplantation. Furthermore, we develop a minimal expression marker panel that discriminates PLT- and multi-lineage (MUL-)HSCs using microfluidics-based single-cell RT-qPCR. We show that both methods detect the age-associated increase in PLT-HSCs, while poly(I-C)-induced chronic inflammation did not alter HSC lineage bias. In contrast, romiplostim treatment increased MUL-HSC prevalence. Finally, using spectral flow cytometry to simultaneously quantify cell cycle and HSC lineage bias, we show that platelet depletion selectively activates PLT-HSCs. Together, these approaches allow accurate isolation of PLT-HSCs and robust quantification of lineage bias under perturbation.

Immune responses to parasite infection involve the increased production of basophils and eosinophils. These two myeloid cell types have key roles in type 2 anti-parasite immunity1 and rely on GATA family transcription factors for their specification2,3. The first committed step in basophil and eosinophil production is generation of basophil-eosinophil-mast cell progenitors (BEMPs) from oligopotent erythroid-primed multipotent progenitors (EMPPs). However, it is not well established how immune responses act on progenitors to initiate type 2 myelopoiesis. Here we show that infection with the helminth Heligmosomoides polygyrus increases EMPP commitment to myeloid fate at the expense of erythropoiesis. Upon infection with H. polygyrus, the IL-33 alarmin accumulated in the bone marrow, causing EMPPs to upregulate the GATA co-factor LMO4 and preferentially differentiate into myeloid cells. LMO4 was sufficient to instruct myeloid fate in EMPPs by interacting with GATA2, displacing the FOG1 co-factor and redistributing GATA binding from megakaryocyte-erythroid-specific to basophil, eosinophil and mast cell (BEM)-specific chromatin. Accordingly, mice carrying a GATA2 mutation that selectively impairs the LMO4-GATA2 interaction were deficient in GATA factor allocation to BEM chromatin, myeloid lineage commitment, basophil and eosinophil production, and parasite control. This identifies LMO4 as an IL-33-regulated master regulator of type 2 myelopoiesis, and transcription factor reallocation as a mechanism of lineage commitment.

Abstract Mouse single-cell ageing studies provide experimentally controlled age contrasts, but using mouse-labelled data to annotate human ageing states is limited by species, donor and assay effects in sparse transcriptomic and chromatin profiles. We developed a cross-species annotation workflow that treats mouse-to-human prediction as a target-validated domain-adaptation problem. The workflow uses orthologue-aligned features, a residual encoder, an age classifier and a species discriminator trained with two-phase adversarial optimisation, and couples prediction with stability-based gene attribution. In haematopoietic stem cells (HSCs), the model achieved held-out human AUROCs of 0.933 in scRNA-seq and 0.953 in scATAC-seq. In an independent CD8+ T-cell scRNA-seq setting, the held-out human AUROC was 0.941. Ablation analyses indicated that residual connections, ELU activation and two-phase training improved predictive performance and attribution stability. Consensus attributions from DeepLIFT, Integrated Gradients and saliency recovered conserved ageing-associated genes with greater cross-species overlap than differential expression alone. In a COVID-19 convalescent cohort, severe disease in younger adults was associated with a higher fraction of CD8+ cells classified as old-like by the pretrained model. These results support a reproducible framework for testing, interpreting and releasing cross-species single-cell ageing models, while highlighting the need for target-domain validation when mouse labels are transferred to human data.

Ex vivo expansion of human hematopoietic stem cells (HSCs) holds promise for overcoming their limited availability, a major barrier to broader clinical application. Although recent advances in culture systems can increase HSC numbers, these conditions frequently impair self-renewal and induce myeloid bias, and the underlying molecular mechanisms remain poorly understood. Here, we performed single-cell multiome sequencing (scMultiome-seq) on human umbilical cord blood-derived CD34⁺ hematopoietic stem and progenitor cells to co-profile transcriptional and epigenetic adaptations within the same cells during ex vivo culture. Our analyses revealed reduced transcriptional and epigenetic HSC signatures, accompanied by markedly increased activity of myeloid-associated transcription factor motifs, providing molecular insight into the functional decline and myeloid bias of cultured HSCs. We further observed substantial functional heterogeneity among phenotypically defined HSCs following culture. To address these limitations, we established a niche-mimetic culture system that integrates intrinsic and extrinsic bone marrow regulatory cues, including pharmacologic inhibition of the m6A reader YTHDF2 using the small molecule Y13-27, a three-dimensional microenvironment, and N-cadherin-mediated adhesion. This condition (3D-NcadP-Y) robustly preserved long-term repopulating capacity. When combined with the self-renewal agonist UM729, the resulting platform (3D-NcadP-Y-UM) uniquely enabled the expansion of serially transplantable long-term HSCs with balanced multilineage potential. scMultiome-seq and cellular analyses demonstrated that this condition preserves transcriptional and epigenetic long-term HSC signatures, maintains multilineage-associated transcription factor motifs, and limits excessive cell-cycle activation. Together, these findings elucidate molecular mechanisms underlying culture-induced HSC dysfunction and establish a niche-mimetic strategy for expanding functional human long-term HSCs while preserving key features of stemness.

Somatic stem cell pools comprise diverse, highly specialized subsets whose individual contribution is critical for the overall regenerative function. In the bone marrow, myeloid-biased hematopoietic stem cells (myHSCs) are indispensable for replenishment of myeloid cells and platelets during inflammatory response but, at the same time, become irreversibly damaged during inflammation and aging. Here we identify an extrinsic factor, semaphorin 4A (Sema4A), which non-cell-autonomously confers myHSC resilience to inflammatory stress. We show that, in the absence of Sema4A, myHSC inflammatory hyper-responsiveness in young mice drives excessive myHSC expansion, myeloid bias and profound loss of regenerative function with age. Mechanistically, Sema4A is mainly produced by neutrophils, signals via a cell surface receptor, plexin D1, and safeguards the myHSC epigenetic state. Our study shows that, by selectively protecting a distinct stem cell subset, an extrinsic factor preserves functional diversity of somatic stem cell pool throughout organismal lifespan. Toghani, Gupte et al. identify semaphorin 4A as a cell-extrinsic factor that protects myeloid-biased hematopoietic stem cells from inflammaging, mainly produced by their progeny—neutrophils—and acting via a negative feedback loop.

Hematopoietic stem cells (HSCs) responsible for blood cell production and their bone marrow regulatory niches undergo age-related changes, affecting immune responses and predisposing individuals to hematologic malignancies. Here, we show that the age-related alterations of the megakaryocytic niche and associated downregulation of platelet factor 4 (PF4) are pivotal mechanisms driving HSC aging. PF4-deficient mice display several phenotypes reminiscent of accelerated HSC aging, including lymphopenia, increased myeloid output, and DNA damage, mimicking physiologically aged HSCs. Remarkably, recombinant PF4 administration restored old HSCs to youthful functional phenotypes characterized by improved cell polarity, reduced DNA damage, enhanced in vivo reconstitution capacity, and balanced lineage output. Mechanistically, we identified low-density lipoprotein receptor and C-X-C motif chemokine receptor 3 as HSC receptors transmitting the PF4 signal, with double knockout mice exhibiting exacerbated HSC aging phenotypes similar to PF4-deficient mice. Furthermore, human HSCs across various age groups also respond to the youthful PF4 signaling, highlighting its potential for rejuvenating aged hematopoietic systems. These findings pave the way for targeted therapies aimed at reversing age-related HSC decline, with potential implications in the prevention or improvement of the course of age-related hematopoietic diseases.

ABSTRACT:Hematopoietic stem cells (HSCs) responsible for blood cell production and their bone marrow regulatory niches undergo age-related changes, affecting immune responses and predisposing individuals to hematologic malignancies. Here, we show that the age-related alterations of the megakaryocytic niche and associated downregulation of platelet factor 4 (PF4) are pivotal mechanisms driving HSC aging. PF4-deficient mice display several phenotypes reminiscent of accelerated HSC aging, including lymphopenia, increased myeloid output, and DNA damage, mimicking physiologically aged HSCs. Remarkably, recombinant PF4 administration restored old HSCs to youthful functional phenotypes characterized by improved cell polarity, reduced DNA damage, enhanced in vivo reconstitution capacity, and balanced lineage output. Mechanistically, we identified low-density lipoprotein receptor and C-X-C motif chemokine receptor 3 as HSC receptors transmitting the PF4 signal, with double knockout mice exhibiting exacerbated HSC aging phenotypes similar to PF4-deficient mice. Furthermore, human HSCs across various age groups also respond to the youthful PF4 signaling, highlighting its potential for rejuvenating aged hematopoietic systems. These findings pave the way for targeted therapies aimed at reversing age-related HSC decline, with potential implications in the prevention or improvement of the course of age-related hematopoietic diseases.

ABSTRACT:Hematopoietic stem cells (HSCs) exhibit significant age-related phenotypic and functional alterations. Although single-cell technologies have elucidated age-related compositional changes, prospective identification of aging-associated HSC subsets has remained challenging. In this study, using clusterin (Clu)-green fluorescent protein (GFP) reporter mice, we demonstrated that Clu expression faithfully marks age-associated myeloid/platelet-biased HSCs throughout life. Clu-GFP expression clearly segregates a novel age-associated HSC subset that overlaps with but is distinct from those previously identified using antibodies against aging maker proteins or reporter systems of aged HSC signature genes. Clu-positive (Clu+) HSCs emerge as a minor population in the fetus and progressively expand with age. Clu+ HSCs display not only an increased propensity for myeloid/platelet-biased differentiation but also a unique behavior in the bone marrow, favoring self-renewal over differentiation into downstream progenitors. In contrast, Clu-negative (Clu-) HSCs exhibit lineage-balanced differentiation, which predominates in the HSC pool during development but becomes underrepresented as aging progresses. Both subsets maintain long-term self-renewal capabilities even in aged mice but contribute differently to hematopoiesis. The predominant expansion of Clu+ HSCs largely drives the age-related changes observed in the HSC pool. Conversely, Clu- HSCs preserve youthful functionality and molecular characteristics into old age. Consequently, progressive changes in the balance between Clu+ and Clu- HSC subsets account for HSC aging. Our findings establish Clu as a novel marker for identifying aging-associated changes in HSCs and provide a new approach that enables lifelong tracking of the HSC aging process.

Hematopoietic stem cells (HSCs) sustain blood cell production throughout the mammalian life span. However, it has become clear that at the single cell level a subset of HSCs is stably biased in their lineage output, and that such heterogeneity may play a key role in physiological processes including aging and adaptive immunity. Analysis of chromatin accessibility, DNA methylation, and histone modifications has revealed that HSCs with different lineage bias exhibit distinct epigenetic traits inscribed at poised, lineage-specific enhancers. This allows for lineage priming without initiating lineage-specific gene expression in HSCs, controlling lineage bias while preserving self-renewal and multipotency. Here, we review our current understanding of epigenetic regulation in the establishment and maintenance of HSC fate decisions under different physiological conditions.

Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well-established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2-activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small-molecule and PROTAC-mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2-activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity.

Hematopoietic stem cells (HSCs) reconstitute multilineage human hematopoiesis after clinical bone marrow (BM) transplantation and are the cells of origin of some hematological malignancies. Although HSCs provide multilineage engraftment, individual murine HSCs are lineage biased and contribute unequally to blood cell lineages. Here, we performed high-throughput single-cell RNA sequencing in mice after xenograft with molecularly barcoded adult human BM HSCs. We demonstrated that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identified platelet-biased and multilineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young and aged BM showed that both the proportion of platelet-biased HSCs and their level of transcriptional platelet priming increase with age. Therefore, platelet-biased HSCs and their increased prevalence and transcriptional platelet priming during aging are conserved features of mammalian evolution.

Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire mutations, most frequently in the DNMT3A and TET2 genes, conferring a competitive advantage through mechanisms that remain unclear. To gain insight into how CH mutations enable gradual clonal expansion, we used single-cell multi-omics with high-fidelity genotyping on human CH bone marrow (BM) samples. Most of the selective advantage of mutant cells occurs within HSCs. DNMT3A- and TET2-mutant clones expand further in early progenitors, while TET2 mutations accelerate myeloid maturation in a dose-dependent manner. Unexpectedly, both mutant and non-mutant HSCs from CH samples are enriched for inflammatory and aging transcriptomic signatures, compared with HSCs from non-CH samples, revealing a non-cell-autonomous effect. However, DNMT3A- and TET2-mutant HSCs have an attenuated inflammatory response relative to wild-type HSCs within the same sample. Our data support a model whereby CH clones are gradually selected because they are resistant to the deleterious impact of inflammation and aging.

A central question in hematopoiesis is how individual blood cell types are specified from multi-potent stem- and progenitor cells, and over the last decade single cell biology has revealed considerable molecular complexity of blood cell progenitors. A key challenge is now to understand the functional properties of this diversity of progenitor cell populations and the cellular and molecular mechanisms by which they are specified.The hematopoietic progenitor compartment is divided into domains that generate GATA-1 expressing (Gata1+) and non-expressing (Gata1–) blood cell types, respectively. We here use Gata1 lineage tracing to show that onset of Gata1 expression coincides with loss of Gata1– lineage potentials. Isolation of progenitors in the process of crossing this fate boundary showed that progenitors committed to a Gata1+ fate are initially oligo-potent, but rapidly resolve into fully lineage-committed megakaryocyte, erythroid and myeloid (here: basophil/mast cell/eosinophil) progenitors, a process that involves dynamic changes to the chromatin access of the corresponding lineage-specific transcription factors (TFs).Detailed analysis of the segregation of myeloid and megakaryocyte/erythroid fates showed that the GATA-2 co-factor LMO4 played a critical role in myeloid lineage commitment by inducing the translocation of GATA-2 to myeloid-specific chromatin. Consequently, a GATA-2 mutant protein selectively lacking LMO4 interaction failed to support myeloid fate. Finally, cytokine modulation of LMO4 expression was able to bias this lineage choice towards myelopoiesis. Therefore, the Gata1+ progenitor domain is hierarchically organised through sequential loss of lineage potentials, and the associated fate choices can be extrinsically controlled by regulation of the expression of lineage-instructive TFs.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Somatic stem cell pools are comprised of diverse, highly specialized subsets whose individual contribution is critical for the overall regenerative function. In the bone marrow, myeloid-biased HSC (myHSC) are indispensable for replenishment of myeloid cells and platelets during inflammatory response but at the same time, become irreversibly damaged during inflammation and aging. Here, we identify an extrinsic factor, Semaphorin 4A (Sema4A), which non cell-autonomously confers myHSC resilience to inflammatory stress. We show that the absence of Sema4A, myHSC inflammatory hyper-responsiveness in young mice drives excessive myHSC expansion, myeloid bias and profound loss of regenerative function with age. Mechanistically, Sema4A is mainly produced by neutrophils, signals via a cell surface receptor Plexin D1 and safeguards myHSC epigenetic state. Our study shows that by selectively protecting a distinct stem cell subset, an extrinsic factor preserves functional diversity of somatic stem cell pool throughout organismal lifespan. ### Competing Interest Statement The authors have declared no competing interest.

Endomucin (EMCN) currently represents the only hematopoietic stem cell (HSC) marker expressed by both murine and human HSCs. Here, we report that EMCN + long-term repopulating HSCs (LT-HSCs; CD150 + CD48- LSK) have a higher long-term multi-lineage repopulating capacity compared to EMCN- LT-HSCs. Cell cycle analyses and transcriptional profiling demonstrated that EMCN + LT-HSCs were more quiescent compared to EMCN- LT-HSCs. Emcn- /- and Emcn +/+ mice displayed comparable steady-state hematopoiesis, as well as frequencies, transcriptional programs, and long-term multi-lineage repopulating capacity of their LT-HSCs. Complementary functional analyses further revealed increased cell cycle entry upon treatment with 5-fluorouracil and reduced granulocyte colony-stimulating factor (GCSF) mobilization of Emcn- /- LT-HSCs, demonstrating that EMCN expression by LT-HSCs associates with quiescence in response to hematopoietic stress and is indispensable for effective LT-HSC mobilization. Transplantation of wild-type bone marrow cells into Emcn- /- or Emcn +/+ recipients demonstrated that EMCN is essential for endothelial cell-dependent maintenance/self-renewal of the LT-HSC pool and sustained blood cell production post-transplant.

Introduction The 2 most frequent somatic mutations in the calreticulin (CALR) gene associated to essential thrombocythemia and myelofibrosis patients are a 52 base-pair (bp) deletion (del52) and a 5 bp insertion (ins5). We previously modeled both types of CALR mutations (CALRmut) in knock-in (KI) mice and observed a more severe megakaryocytic (MK) phenotype with CALRdel52 than with CALRins5, including a strong amplification and competitive advantage of CALRdel52 hematopoietic stem cells (HSC). Both types of CALRmut acquire a new C-terminal tail that stabilizes their interaction to the thrombopoietin receptor, stimulating its oncogenic signaling at the cell surface. Moreover, CALRmut lose their endoplasmic reticulum (ER) retrieval KDEL motif leading to their secretion, ER stress and activation of the unfolded protein response (UPR). However, the disease-initiating cells within the HSC compartment have not been identified and the precise mechanism of action of CALRmut remains incompletely understood. These results are essential to enable effective therapeutic targeting. Methods To analyze the effects of CALRmut in HSC, we used a combination of single cell techniques (scRNAseq and mass cytometry) and KI mice expressing the murine CALRdel52 or CALRins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase. These KI mice were crossed with transgenic mice expressing the GFP under the control of the von Willebrand factor (Vwf) promoter. Results We combined scRNAseq analyses of mouse LSK progenitors and results from a trajectory inference algorithm on mass cytometry data. We observed on a UMAP plot an expansion of HSC (1.8-fold) and multipotent progenitors MPP2-3 (1.5-fold) with CALRdel52 and MPP1 (1.2-fold) with CALRins5 at the expense of lymphoid-primed MPP4 (4.4-fold and 1.8-fold less with CALRdel52 and CALRins5, respectively). Consistent with the stronger amplification of CALRdel52 HSC, we found a significant upregulation of Ki-67 in these cells and a higher proliferation index compared to wild-type (WT) and CALRins5 HSC (4.72 vs 3.22 and 3.45). We also detected a significant activation of eiF2a from the PERK branch of the UPR in CALRdel52 but not in CALRins5 HSC, which decreased to WT level upon MK differentiation. Interestingly, we observed an upregulation of vWF gene expression in CALRdel52 HSC, indicating a platelet/myeloid bias. Differential analysis between vWF-positive (vWF+) HSC, that are at the top of hematopoiesis, and vWF-negative (vWF-) HSC pointed to the upregulation of a UPR signature in the vWF+ context that was exacerbated by CALRdel52. Using capillary electrophoresis and qPCR, we confirmed in CALRdel52 KI mice carrying the Vwf promoter-GFP construct that eiF2a was predominantly phosphorylated and activated in vWF+vs vWF- HSC. The function of these sorted vWF- or vWF+ HSC was analyzed after engraftment in lethally-irradiated recipient mice. Both groups of mice developed thrombocytosis, but mice engrafted with vWF+CALRmut HSC had a more pronounced disease phenotype, particularly with CALRdel52 (n=7-10; 4,893±299 vs 3,283±398 x103/mL platelets at 20 weeks after CALRdel52 expression in vWF+vs vWF- HSC and 2,180±112 vs 1,462±104 for CALRins5). Secondary and tertiary bone marrow transplantations (n=5-10) undoubtedly identified vWF+CALRmut HSC rather than vWF- HSC as the disease-initiating cells. The vWF+ HSC can directly differentiate into MK via a biased route or in a stepwise manner via the generation of vWF- HSC. CALRmut, especially CALRdel52, led to a significant expansion of vWF+ HSC without exacerbating their platelet bias. Notably, both CALRmut conferred platelet-biased properties to the vWF- HSC while retaining their lymphoid potential. Conclusions In conclusion, using CALRmut mouse models, we demonstrate that thrombocytosis is initiated from the amplification of the disease-initiating platelet-biased vWF+ HSC and the increased capacity of vWF- HSC to give platelets. The differential amplification of CALRdel52vsCALRins5 HSC might in part be explained by the activation of the PERK/eiF2a branch of the UPR in the vWF+CALRdel52 HSC sub-population. These results highlight a new cell of origin and a mechanism of action of CALRdel52 in HSC that could be targeted as part of a therapeutic approach.