Haematopoietic stem cells (HSCs) reside in specialized microenvironments, referred to as niches, and the classical model suggests that HSC numbers are predominantly determined by the niche size1-5. However, the vast excess of niche cells relative to HSCs challenges this perspective. To rigorously define the role of niche size in regulating HSC numbers, we developed a femur-transplantation system, enabling us to increase available HSC niches. Notably, the addition of niches did not alter the total HSC numbers in the body, suggesting the presence of a systemic mechanism that limits HSC numbers. Additionally, HSC numbers in transplanted wild-type femurs did not exceed physiological levels when HSCs were mobilized from defective endogenous niches to the periphery, indicating that HSC numbers are constrained at the local level as well. The notion of dual restrictions at systemic and local levels was further supported by other experimental approaches, including parabiosis and non-conditioned transfer of HSCs after bone transplantation. Moreover, we found that thrombopoietin has a pivotal role in determining the total number of HSCs in the body, even in the context of increased niche availability. Our study redefines key principles underlying HSC number regulation, providing insights into this critical biological process.

Bone marrow mesenchymal stromal cells (MSCs) are a major source of secreted factors that control hematopoietic stem and progenitor cell (HSPC) function. We previously reported the generation of revitalized MSCs (rMSCs), which more effectively support HSPCs in culture. In a secretome screen using rMSCs, we identified semaphorin 3A (SEM3A) as a secreted factor upregulated as part of a pro-inflammatory signature that may contribute to HSPC expansion by rMSCs. We show that recombinant SEM3A acts directly on HSPCs to inhibit their cycling ex vivo . Analysis of a SEM3A loss of function mutation in vivo revealed hematopoietic progenitor expansion and accelerated recovery after myeloablation, consistent with a role for SEM3A in regulating HSPCs at steady state and during hematopoietic stress. This work highlights proteomic screening using rMSCs as a method to identify novel secreted niche factors and uncovers a novel role for SEM3A in controlling HSPC proliferation in stress hematopoiesis. Summary Borger et al. characterize the secretome of revitalized bone marrow stromal cells and identify a novel role of the protein semaphorin 3A in regulating hematopoietic stem and progenitor cell proliferation in steady state and stress conditions. ### Competing Interest Statement P.S.F. served as a consultant for Pfizer, received research funding from Ironwood Pharmaceuticals outside the submitted work, and was a shareholder of Cygnal Therapeutics. K.G. has received research funding from ADC Therapeutics and iOnctura outside the submitted work. All other authors declare no competing interests. The raw LC-MS/MS data, process report files, and extracted peptide features for the pSILAC experiments have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository under the accession code PXD065939 . The raw LC-MS/MS data and annotated spectra for the unlabeled proteomic experiments are available under the accession code PXD065232 . Code used for analysis and visualization of transcriptomic and proteomic data is available upon reasonable request. National Institutes of Health, DK056638, DK130895, F30HL154749, S10OD026833, S10OD032169, P30CA013330 New York State Department of Health, https://ror.org/04hf5kq57, C029154, C029570 National Institute of General Medical Sciences, https://ror.org/04q48ey07, 5T32GM007491, 5T32GM007288

NPM1-mutated acute myeloid leukemia (AML) is defined by aberrant cytoplasmic localization of the mutant NPM1c protein, and therapeutic strategies targeting this specific disease remain limited. Here, we identify TTLL4, a mono-glutamate glutamyltransferase, as a selective vulnerability in NPM1c AML. TTLL4 catalyzes post-translational hyper-glutamylation of NPM1c at E126, stabilizes its cytoplasmic localization and promotes a differentiation block in leukemic cells. Multiple genetic TTLL4 inactivation approaches in human NPM1c-mutant cell lines reduce NPM1c glutamylation, trigger myeloid differentiation, and impair proliferation. Transcriptomic analyses show that TTLL4 knockdown pheno-copies NPM1c degradation and aligns with KMT2A and XPO1-targeted gene expression programs. Furthermore, Ttll4 knockout significantly prolonged survival in an NPM1c/NRAS-driven mouse AML model and promoted differentiation. We identify a small molecule, EN7, that selectively inhibits TTLL4 and recapitulates these phenotypes in NPM1c+ cells. These findings identify glutamylation as a new axis of leukemic regulation and highlight TTLL4 as a druggable epigenetic regulator in NPM1c AML. ### Competing Interest Statement The authors declare that they have pending intellectual property patent protection on TTLL4 as a target of chemical perturbation.

Mesenchymal stem and progenitor cells (MSPCs) are a major component of the hematopoietic stem cell (HSC) niche, supporting HSCs in part via secretion of protein factors. Cultured primary MSPCs cells lose the ability to support HSCs in culture, in part due to alterations in the MSPC secretome. Previous work by our lab identified five transcription and signaling factors, dubbed KOXII factors, that can revitalize murine MSPCs to restore HSC support ex vivo. To identify additional factors that mediate this improved HSC support, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize the secretome of both KOXII-transduced MSPCs and control MSPCs (C-MSPCs), both in isolation and in co-culture with hematopoietic cells. We found that co-culture with hematopoietic cells dramatically increased the secretory activity of MSPCs, suggesting a role of hematopoietic cells in instructing MSPC behavior. This approach also revealed that semaphorin 3a (SEM3A) was dramatically upregulated in the conditioned media of KOXII MSPCs compared to control MSPCs (C-MSPCs). We found that recombinant SEM3A directly supports hematopoietic progenitor survival and quiescence, leading to progenitor expansion. Analysis of published single cell RNAseq datasets demonstrates that SEM3A is not robustly expressed by bone marrow MSPCs at steady state. However, qPCR performed on sorted niche cells following 5-fluorouracil injection showed upregulation of SEM3A in MSPCs under stress. Therefore, our ongoing studies are exploring the roles of SEM3A in the regulation of HSC quiescence under stress conditions. In summary, secretome analysis of KOXII-MSPCs has identified a previously unappreciated role for MSPC-derived SEM3A in the regulation of HSCs and progenitors, adding to emerging evidence for the semaphorin family playing important roles in the regulation of hematopoietic progenitors.

Since Schofield proposed the niche model in the 1970s, it has been widely believed that the number of hematopoietic stem cells (HSCs) is determined by the niche size. We and others have identified several components of the HSC niche, including Nestin-GFP + mesenchymal stem cells (MSCs). However, the number of defined niche cells vastly exceeds that of HSCs, raising the question about the actual regulatory mechanisms governing HSC numbers. To address this question, we initially established a mathematical model of niche availability and occupancy. The model predicted that an increase in available niches initially leads to a corresponding rise in the number of occupying HSCs, which aligns with the concept of local, niche availability-driven restriction on HSC numbers. Interestingly, our model also suggested that this increase in HSC numbers saturates when the number of available niches reaches a critical level. One potential mechanism for this saturation is that HSC numbers may also be regulated at the systemic level, with total HSC numbers in the body being restricted. To experimentally test this model and hypothesis, we developed a bone transplantation technique that allows us to increase the number of available HSC niches. When a single femur (referred to as a graft) was transplanted per mouse (called a host), we observed the progressive regeneration of bone marrow (BM) stroma and hematopoietic recovery in the grafts. While the number of HSCs (Lin -Sca-1 +c-Kit +CD150 +CD48 -CD34 -) in the grafts was still 41.7 ± 7.60% of that in the host femurs at 5 months after transplantation (n = 8, P = 0.0002), they were comparable at 3 months after transplantation when granulocyte colony-stimulating factor (G-CSF) was administered to hosts at 1 month after transplantation (n = 8, P = 0.660). By utilizing Nestin-GFP transgenic mice and a CD45 congenic system, we found that MSCs in grafts are derived from the graft, while hematopoietic cells are derived from the host. Given that a single femur harbors only 8.3 ± 0.440% of HSCs in the total body (n = 11), 6 femurs were transplanted per mouse, followed by G-CSF administration, to assess the impact of increased HSC niches on total HSC numbers. Strikingly, HSC numbers per host femur and graft femur were lower than those per femur from sham-operated mice (n = 7-48, P = 0.012). Moreover, we found that the sum of HSC numbers in the bone transplantation host and graft bones was equivalent to that in the sham-operated group (n = 7-8, P = 0.364). Total HSC numbers were also maintained when 6 femurs from wild-type (WT) mice were transplanted into Cdh2 (N-Cadherin)-CreER; Cxcl12fl/fl mice, which have an impaired ability to retain HSCs in the BM (n = 8, P = 0.711). These results indicate that the addition of normal niches does not alter total HSC numbers in the body, regardless of whether the endogenous niche is functional, supporting the conclusion that HSC numbers are indeed restricted at the systemic level (Figure 1A). To determine whether HSC numbers are also limited at the local level, as suggested by our mathematical model, we transplanted a single WT femur to Cxcl12fl/fl or Cdh2-CreER; Cxcl12fl/fl mice. We hypothesized that implanting only one femur would have a minimal impact on the total BM, and therefore HSC numbers in the grafts transplanted to Cxcl12fl/fl mice would reach the physiological level. We reasoned that if there is no local restriction on HSC numbers, there would be more HSCs in the WT grafts transplanted to Cdh2-CreER; Cxcl12fl/fl mice compared with those implanted to Cxcl12fl/fl mice. As expected, HSC numbers per WT graft femur in Cxcl12fl/fl mice were comparable to those per femur of sham-operated Cxcl12fl/fl mice (corresponding to the physiological level) (n = 8, P = 0.433). However, CXCL12 deficiency in the host BM did not affect HSC numbers per WT graft femur (n = 8, P = 0.986). Importantly, the sum of HSC numbers in the host body and the graft femur of Cdh2-CreER; Cxcl12fl/fl recipient mice was lower than that of sham-operated Cxcl12fl/fl mice (n = 8, P = 0.003), indicating that total HSC numbers in the former mice are below the systemic limit. Collectively, these data suggest that the failure to increase HSC numbers per WT graft in Cdh2-CreER; Cxcl12fl/fl hosts is, at least in part, due to a local restriction on HSC numbers (Figure 1B). Our study demonstrates that HSC numbers are restricted at both systemic and local levels, thereby rewriting the long-standing model for the regulation of HSC numbers.

Haematopoietic stem cells (HSCs) reside in specialized microenvironments, also referred to as niches, and it has been widely believed that HSC numbers are determined by the niche size alone 1-5 . However, the vast excess of the number of niche cells over that of HSCs raises questions about this model. We initially established a mathematical model of niche availability and occupancy, which predicted that HSC numbers are restricted at both systemic and local levels. To address this question experimentally, we developed a femoral bone transplantation system, enabling us to increase the number of available HSC niches. We found that the addition of niches does not alter total HSC numbers in the body, regardless of whether the endogenous (host) niche is intact or defective, suggesting that HSC numbers are limited at the systemic level. Additionally, HSC numbers in transplanted wild-type femurs did not increase beyond physiological levels when HSCs were mobilized from defective endogenous niches to the periphery, indicating that HSC numbers are also constrained at the local level. Our study demonstrates that HSC numbers are not solely determined by niche availability, thereby rewriting the long-standing model for the regulation of HSC numbers.

Host microbiota crosstalk is essential for the production and functional modulation of blood-cell lineages. Whether, and if so how, the microbiota influences hematopoietic stem cells (HSCs) is unclear. Here, we show that the microbiota regulates HSC self-renewal and differentiation under stress conditions by modulating local iron availability in the bone marrow (BM). In microbiota-depleted mice, HSC self-renewal was enhanced during regeneration, while the commitment toward differentiation was dramatically compromised. Mechanistically, microbiota depletion selectively impaired the recycling of red blood cells (RBCs) by BM macrophages, resulting in reduced local iron levels without affecting systemic iron homeostasis. Limiting iron availability in food (in vivo) or in culture (ex vivo), or by CD169+ macrophage depletion, enhanced HSC self-renewal and expansion. These results reveal an intricate interplay between the microbiota, macrophages, and iron, and their essential roles in regulating critical HSC fate decisions under stress.

Haematopoietic stem cells (HSCs) home to the bone marrow via, in part, interactions with vascular cell adhesion molecule-1 (VCAM1)1–3. Once in the bone marrow, HSCs are vetted by perivascular phagocytes to ensure their self-integrity. Here we show that VCAM1 is also expressed on healthy HSCs and upregulated on leukaemic stem cells (LSCs), where it serves as a quality-control checkpoint for entry into bone marrow by providing ‘don’t-eat-me’ stamping in the context of major histocompatibility complex class-I (MHC-I) presentation. Although haplotype-mismatched HSCs can engraft, Vcam1 deletion, in the setting of haplotype mismatch, leads to impaired haematopoietic recovery due to HSC clearance by mononuclear phagocytes. Mechanistically, VCAM1 ‘don’t-eat-me’ activity is regulated by β2-microglobulin MHC presentation on HSCs and paired Ig-like receptor-B (PIR-B) on phagocytes. VCAM1 is also used by cancer cells to escape immune detection as its expression is upregulated in multiple cancers, including acute myeloid leukaemia (AML), where high expression associates with poor prognosis. In AML, VCAM1 promotes disease progression, whereas VCAM1 inhibition or deletion reduces leukaemia burden and extends survival. These results suggest that VCAM1 engagement regulates a critical immune-checkpoint gate in the bone marrow, and offers an alternative strategy to eliminate cancer cells via modulation of the innate immune tolerance. Pinho et al. show that VCAM1 and MHC-I cooperate to provide a ‘don’t-eat-me’ signal that prevents haematopoietic stem cells clearance by mononuclear phagocytes, and also that VCAM1 can be hijacked by cancer cells to escape innate immune surveillance.

Hematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors (Scf, Cxcl12, Vcam1 and Angpt1) by Nestin-GFP+ mesenchymal-derived stem cells (MSCs) is downregulated upon culture and lose its effect of maintaining HSC in vitro, suggesting that transcriptional rewiring may contribute to this reduced potential in cultured MSCs. To gain further insight, we searched RNA sequencing data for transcriptional regulators that were highly expressed in Nestin-GFP+ stroma, revealing 40 potential candidates. We compared the expression of these genes by real-time quantitative PCR (qPCR) in freshly isolated Nestin-GFP+ or Nestin-GFP- BM CD45-Ter119-CD31- cells, with that of cultured Nestin-GFP+ stroma. These analyses yielded 28 candidate genes after the elimination of 12 genes due to non-specific expression or lack of downregulation after culture. We cultured stromal cells isolated from Scf-GFP knock-in mice in which GFP expression reflects endogenous Scf mRNA synthesis. Upon culture, GFP expression was rapidly downregulated in these cells, demonstrating the potential of using GFP to screen for factors capable of revitalizing niche activity in cultured MSCs. We generated lentiviral vectors expressing 28 selected genes and transduced the viral mixture into cultured stromal cells derived from Scf-GFP mice. Five days after transduction, we observed re-emergence of GFP+ cells and these GFP+ cells were sorted and plated in limiting dilutions to isolate single cell-derived clones. Using this approach, we generated 16 independent GFP+ single cell-derived clones. To determine the specific combination of genes that enables cultured stromal cells to regain their capacity to maintain and expand HSCs in vitro, lineage-negative (Lin-) BM cells were co-cultured with each single cell-derived clone or control stroma. Thus, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, and Irf7; KOXII) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs in vitro showed higher repopulation capacity and enabled lethally irradiated recipient mice to survive better. Competitive reconstitution assays revealed 7-fold expansion of functional HSCs by rMSCs. Moreover, rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. To investigate the revitalization mechanism, we performed ATAC-seq in freshly sorted Scf-GFP- CD45-Ter119-CD31- cells, Scf-GFP+ CD45-Ter119-CD31- cells, rMSCs and control vector-transduced stroma. We found that revitalization of MSCs led to 9,623 peaks of open chromatin in rMSCs when compared to control MSCs. Of these, 626 open peaks were also detected in freshly isolated Scf-GFP+ cells when compared to Scf-GFP- cells. Motif analyses of the sequence at these 626 peaks revealed that myocyte enhancer factor 2c (Mef2c) was among the most significantly enriched transcription regulators. Mef2c was also expressed at high levels in both rMSCs and freshly isolated Scf-GFP+ cells compared to control cultured MSCs and freshly isolated Scf-GFP- cells by RNA-seq and real-time qPCR. To evaluate the role of Mef2c in rMSCs, we knocked down Mef2c in rMSCs by short hairpin RNA lentiviral transduction (shMef2c). We found that the expression of niche factors (Scf, Cxcl12 and Vcam1) was reduced in shMef2c-transduced compared to parental rMSCs. In addition, shMef2c transduced-rMSCs exhibited reduced (by 43%) capacity to expand HSCs in co-culture compared to shCntrl transduced-rMSCs. These results suggest a role for Mef2c as a downstream effector mediating MSC revitalization. We are now exploring the method to make these rMSCs to form new niches in vivo. Our results suggest that combination of KOXII genes are able to fully restore the niche activity in MSCs ex vivo and establish a new platform that provides critical insight in the regulatory network of the HSC niche leading to the basis toward the engineering of supportive niches for curative cell therapies. Disclosures Wei: Albert Einstein College of Medicine, Inc: Patents & Royalties. Frenette:Albert Einstein College of Medicine, Inc: Patents & Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.

Haematopoietic stem cells (HSCs) are maintained by bone marrow niches in vivo 1 , 2 , but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP + mesenchymal-derived stromal cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified five genes encoding transcription factors ( Klf7 , Ostf1 , Xbp1 , Irf3 and Irf7 ) that restored HSC niche function in cultured bone marrow-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed an approximately sevenfold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. Analysis of the reprogramming mechanisms uncovered a role for myocyte enhancer factor 2c ( Mef2c ) in the revitalization of MSCs. These results provide insight into the transcriptional regulation of the niche with implications for stem cell-based therapies.

Hematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stem cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, Irf7) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed 7-fold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. We have also identified Mef2c as an important transcription factor downstream of KOXII. Our results suggest that all five KOXII genes are necessary to fully restore the niche activity in MSCs ex vivo. Some of these genes may act singly, perhaps by preventing MSC differentiation or driving expansion of hematopoietic growth factors. Our results thus establish a new platform that provides critical insight in the regulatory network of the HSC niche and forms the basis toward the engineering of supportive niches for curative cell therapies.

Hematopoietic stem cells (HSCs) home to the bone marrow (BM) via, in part, the interactions with Vascular Cell Adhesion Molecule-1 (VCAM1). Upon migrating into the BM, HSCs are vetted by perivascular phagocytes to ensure their self-integrity. Here, we show that VCAM1 is also expressed on healthy Lineage− c-Kit+ Sca1+ CD48− CD150+ HSCs and upregulated on acute myeloid leukemia (AML) cells where it serves as a quality-control checkpoint for entry into BM by providing 'don't-eat-me' stamping in the context of major histocompatibility complex (MHC) class-I presentation. Using a line in which HSCs are hemizygous for MHC (H-2b/H-2q) and Vcam1 can be conditionally deleted by breeding Vcam-1 floxed (Vcam1fl/fl) mice with a Csf1r-iCre transgenic line (referred to as Vcam1cKO), we find that Vcam1 deletion in HSCs leads to striking defects in hematopoietic recovery when donor and recipients are mismatched (donor H-2b/q; recipient H-2b). Remarkably, wild-type donor BM cells with haplotype-mismatched genotype exhibit engraftment and survival similar to their syngeneic (H-2b) Vcam1fl/fl (control) and Vcam1cKO counterparts. In vivo phagocytic assays show that Vcam1cKO;H-2b/q HSCs are phagocytosed by host H-2b immune phagocytes whereas Vcam1-sufficient H-2b/q HSCs are not targeted. Clearance is not dependent on irradiation-induced damage since the same phenotype is observed in parabiotic mice where Vcam1cKO;H-2b/q HSCs are not able to engraft their wild-type (H-2b) parabiont partner. Mechanistically, we show that MHC-mismatched HSCs are recognized at least in part by paired Ig-like receptor-B (PIR-B), a negative regulator of immune cells upon recognition of MHC-I molecules, expressed on α4β1+ murine phagocytic myeloid cells. Transplantation assays revealed that in absence of PIR-B inhibitory signals, Vcam1 deletion leads to significant reductions (~60%) in the early engraftment of syngeneic Vcam1cKO;H-2b/b cells compared to Vcam1fl/fl;H-2b/b cells, suggesting that the absence of VCAM1 also promotes cell clearance by syngeneic Pirb-/- phagocytes. That VCAM1 expression can provide innate immune tolerance indicates that this pathway may be of use for cancer cells. Indeed, alterations in VCAM1 expression has been reported in various cancer cell types, including AML. To evaluate the effect of genetic Vcam1 deletion and MHC-mismatch on AML progression, we have transduced Vcam1cKO;H-2b/q and Vcam1fl/fl;H-2b/q cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, analysis of primary AML recipient BM reveals a marked reduction (>99%) of phenotypic Vcam1cKO leukemic stem cells compared to Vcam1fl/fl control. Accordingly, the survival of secondary AML recipient mice is significantly prolonged in mice harboring Vcam1cKO AML cells relative to Vcam1fl/fl AML. Leukemia progression is markedly enhanced by phagocyte depletion (using clodronate liposomes), and the engraftment defect of Vcam1cKO AML cells is completely rescued, indicating an important role for phagocytes in the establishment of Vcam1-deficient AML. Analysis of The Cancer Genome Atlas (TCGA) database reveals that high VCAM1 expression is associated with the poor prognosis of patients with AML (P=0.03). In addition, VCAM1 is significantly overexpressed in Lineage− CD34+ CD38− CD90− short-term repopulating HSCs from sorted human AML BM samples. VCAM1 overexpression in human AML MOLM13 cancer cells promotes disease progression and significantly (P=0.005) shorter survival. Furthermore, VCAM1 blockade using novel monoclonal antibodies after implantation of primary human AML into immunodeficient mice blunts disease progression, and extends survival of mice. Altogether, these results suggest that VCAM1 engagement serves as a quality-control checkpoint for entry into BM by providing 'don't-eat-me' stamping, allowing HSCs to migrate and engraft, and offers a novel strategy to eliminate cancer cells via modulation of the innate immune tolerance. Disclosures Wei: Albert Einstein College of Medicine, Inc: Patents & Royalties. Verma:Janssen: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding. Frenette:Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents & Royalties; Pfizer: Consultancy.

Induced pluripotent stem cells (iPSCs) can be derived from small and readily available tissue samples, most often skin or blood samples. But of the various diseases that have been modeled, one class of inherited diseases has been a particular focus of iPSC-based research: lysosomal storage diseases (LSDs). LSDs are a class of approximately 50 metabolic disorders caused by inherited deficiencies in various lysosomal proteins. The majority of LSDs are the result of mutations in metabolic enzymes active in the lysosomal lumen, although several LSDs are caused by defects in lysosomal transport or vesicular trafficking. Gaucher Disease (GD) was among the first diseases for which iPSCs were generated. Not long after the initial announcement of the development of human iPSCs, Park et al. Several groups have also further characterized GD iPSC-derived dopaminergic neurons in attempts to link decreased GCase activity with the observed predisposition toward Parkinson disease and related disorders.

Induced pluripotent stem cells (iPSCs) have provided new opportunities to explore the cell biology and pathophysiology of human diseases, and the lysosomal storage disorder research community has been quick to adopt this technology. Patient-derived iPSC models have been generated for a number of lysosomal storage disorders, including Gaucher disease, Pompe disease, Fabry disease, metachromatic leukodystrophy, the neuronal ceroid lipofuscinoses, Niemann-Pick types A and C1, and several of the mucopolysaccharidoses. Here, we review the strategies employed for reprogramming and differentiation, as well as insights into disease etiology gleaned from the currently available models. Examples are provided to illustrate how iPSC-derived models can be employed to develop new therapeutic strategies for these disorders. We also discuss how models of these rare diseases could contribute to an enhanced understanding of more common neurodegenerative disorders such as Parkinson's disease, and discuss key challenges and opportunities in this area of research.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylceramideladen macrophages resulting from impaired digestion of aged erythrocytes or apoptotic leukocytes. Studies of macrophages from patients with type 1 Gaucher disease with genotypes N370S/N370S, N370S/L444P or N370S/c.84dupG revealed that Gaucher macrophages have impaired efferocytosis resulting from reduced levels of p67(phox) and Rab7. The decreased Rab7 expression leads to impaired fusion of phagosomes with lysosomes. Moreover, there is defective translocation of p67(phox) to phagosomes, resulting in reduced intracellular production of reactive oxygen species. These factors contribute to defective deposition and clearance of apoptotic cells in phagolysosomes, which may have an impact on the inflammatory response and contribute to the organomegaly and inflammation seen in patients with Gaucher disease.