_:b606264085 . _:b606264029 "7"^^ . . _:b51921533 . _:b606264432 "2"^^ . _:b606264026 "7"^^ . _:b606264435 "2"^^ . _:b606264027 "7"^^ . _:b606264434 "2"^^ . _:b606264305 . _:b606264122 . _:b606264024 "7"^^ . . _:b606264445 "2"^^ . _:b606264025 "7"^^ . . _:b606264404 . _:b606264444 "2"^^ . _:b606264038 "6"^^ . . _:b606264447 "2"^^ . _:b606264224 . _:b606264039 "6"^^ . _:b606264446 "2"^^ . _:b606264036 "6"^^ . _:b606264242 . _:b606264057 . _:b606264441 "2"^^ . . _:b606264037 "6"^^ . _:b606264440 "2"^^ . _:b606264034 "6"^^ . . _:b606264100 . _:b606264443 "2"^^ . _:b606264035 "6"^^ . _:b606264356 . _:b606264442 "2"^^ . _:b606264032 "6"^^ . . _:b606264180 . . _:b606264033 "6"^^ . _:b606264037 . _:b606264433 . _:b606264046 "6"^^ . . . _:b51921539 "These results are consistent with mouse models that drive expression of PML-RARA from a myeloid lineage-restricted promoter resulting in an APL-like disease >>40<<,41. The absence of PML-RARA translocation in human HSC obtained from APL patient samples is surprising given that PML-RARA on its own induced myeloid commitment rather than self-renewal in hematopoietic progenitors such as GMP 42. In" . . . _:b606264044 "6"^^ . _:b606264297 . _:b606264147 . _:b606264392 . . _:b606264449 "2"^^ . _:b606264047 "5"^^ . _:b606264045 "6"^^ . . _:b606264273 . _:b606264448 "2"^^ . _:b606264042 "6"^^ . _:b606264027 . _:b606264451 "2"^^ . _:b606264043 "6"^^ . _:b606264189 . . . _:b606264450 "2"^^ . _:b606264040 "6"^^ . . _:b606264041 "6"^^ . _:b51921527 "neoplasm (MPN) commonly associated with JAK2V617F and calreticulin (CALR) mutations 25-28 that has shown only limited engraftment with transplantation of large numbers of patient-derived CD34+ cells in conventional xenograft models >>29<<-31. We examined the ability of humanized ossicles to facilitate the engraftment of MF (see Supplementary Table 6 for patient sample information) through the direct intraossicle injection of FACS-purified CD34+ cells." . . . _:b606264091 . _:b606264448 . _:b606264449 . _:b606264053 "5"^^ . _:b606264450 . _:b606264451 . _:b606264138 . . _:b606264052 "5"^^ . _:b51921516 "1e left panel). Daily administration of an anabolic dose of human parathyroid hormone (40 \u03BCg/kg) for 28 days after BM-MSC transplantation resulted in a significant increase in the size of the humanized ossicles (Supplementary Fig. 1f).>>18<<,19 In order to confirm the human origin of ossicle bone and stromal niche elements, BM-MSC were transduced with lentivirus to stably express green fluorescent protein (GFP)." . _:b51921547 . . _:b606264164 . . _:b606264307 . . . _:b51921541 "The absence of PML-RARA translocation in human HSC obtained from APL patient samples is surprising given that PML-RARA on its own induced myeloid commitment rather than self-renewal in hematopoietic progenitors such as GMP >>42<<. In contrast to a recently published report demonstrating establishment of APL from transduced cord blood-derived CMP, we did not identify PML-RARA fusion events in FACS-purified CMP from primary patient samples 43. How PML-RARA initiates" . _:b606264055 "5"^^ . _:b606264062 . . . _:b606264279 . _:b606264054 "5"^^ . _:b606264049 "5"^^ . . _:b606264019 . _:b606264048 "5"^^ . _:b606264275 . . . _:b606264051 "5"^^ . _:b606264325 . . _:b606264050 "5"^^ . _:b606264233 . _:b606264324 . _:b606264061 "5"^^ . . _:b606264327 . _:b606264257 . _:b606264060 "5"^^ . _:b51921527 . . _:b606264326 . _:b51921529 . _:b606264063 "5"^^ . _:b606264321 . . _:b606264062 "5"^^ . _:b606264320 . _:b606264057 "5"^^ . . _:b606264323 . _:b606264056 "5"^^ . _:b606264142 . _:b606264390 . _:b606264322 . _:b606264059 "5"^^ . _:b606264414 . _:b606264333 . _:b606264112 . _:b606264277 . _:b606264009 . _:b606264058 "5"^^ . _:b606264332 . . _:b606264069 "4"^^ . _:b606264174 . _:b606264335 . . _:b606264068 "5"^^ . _:b606264334 . _:b606264064 . _:b606264071 "4"^^ . _:b606264309 . _:b606264389 . _:b606264329 . _:b606264070 "4"^^ . _:b606264056 . . _:b606264328 . . _:b606264065 "5"^^ . _:b606264331 . _:b606264025 . _:b606264141 . . _:b606264064 "5"^^ . _:b606264330 . _:b606264067 "5"^^ . . _:b606264341 . _:b606264066 "5"^^ . _:b606264340 . _:b606264077 "4"^^ . _:b606264119 . _:b606264343 . _:b606264076 "4"^^ . _:b606264342 . . . _:b606264079 "4"^^ . _:b606264063 . _:b606264366 . _:b51921561 . _:b606264337 . _:b606264134 . _:b606264313 . . _:b606264078 "4"^^ . _:b606264132 . _:b606264336 . _:b606264073 "4"^^ . . _:b606264339 . _:b606264072 "4"^^ . _:b606264359 . _:b606264254 . _:b606264338 . _:b606264075 "4"^^ . _:b606264349 . _:b606264327 . _:b606264074 "4"^^ . _:b606264324 . . _:b606264325 . _:b51921538 "chromosomal translocation is a matter of debate24 with in vitro studies implicating immature compartments able to produce myeloid and erythroid output 36, and alternative reports supporting APL arises from committed myeloid progenitors.>>37<<-39 Here, we report successful fulminant engraftment of PML-RARA-positive human APL cells in humanized ossicles, and identification of APL-initiating cells in an aberrant CD34-/lo GMP-like population." . _:b606264326 . _:b606264348 . _:b606264327 . _:b606264085 "4"^^ . _:b606264320 . . _:b606264089 . _:b606264321 . . _:b606264322 . _:b606264351 . . _:b606264323 . _:b606264084 "4"^^ . _:b606264197 . _:b606264332 . _:b606264333 . _:b606264334 . _:b51921509 "Additionally, many other hematopoietic neoplasms do not engraft in the currently available mouse strains, as transplantation of MDS, MPN, or multiple myeloma has met with very limited success >>10<<-12, although in myelodysplastic syndrome (MDS), a recent study utilized a modified NSG engraftment assay through the co-transplantation of mesenchymal stromal cells (MSC) with HSC to identify MDS-initiating cells 13." . _:b606264350 . _:b606264335 . _:b606264087 "4"^^ . _:b606264379 . _:b606264328 . _:b606264329 . _:b51921553 . _:b606264330 . _:b606264345 . _:b606264331 . _:b606264086 "4"^^ . _:b606264340 . _:b606264040 . _:b606264341 . _:b606264342 . _:b606264344 . _:b606264343 . _:b606264081 "4"^^ . _:b606264336 . _:b606264337 . _:b606264338 . _:b606264394 . _:b606264347 . _:b606264339 . _:b606264080 "4"^^ . _:b606264348 . _:b606264349 . . _:b606264350 . _:b606264346 . _:b606264038 . _:b606264351 . _:b606264083 "4"^^ . _:b606264344 . _:b606264072 . . _:b606264345 . _:b606264346 . _:b606264357 . _:b606264039 . _:b606264347 . _:b606264082 "4"^^ . _:b606264356 . _:b606264357 . _:b606264358 . _:b606264356 . _:b606264359 . _:b606264093 "4"^^ . _:b606264352 . . _:b606264353 . _:b606264354 . _:b606264359 . _:b606264355 . _:b606264437 . _:b606264092 "4"^^ . _:b606264364 . _:b606264365 . _:b606264239 . _:b606264366 . _:b606264358 . _:b606264367 . _:b606264095 "4"^^ . _:b51921555 . _:b606264360 . _:b606264361 . _:b606264362 . _:b606264353 . _:b606264092 . _:b606264363 . _:b606264094 "4"^^ . _:b606264372 . _:b606264015 . _:b606264373 . . _:b606264352 . _:b606264374 . _:b606264375 . _:b51921539 . _:b606264089 "4"^^ . _:b606264368 . _:b606264369 . . _:b606264355 . _:b606264370 . _:b606264371 . _:b606264088 "4"^^ . _:b606264380 . _:b606264381 . _:b606264382 . _:b606264354 . _:b606264249 . _:b606264383 . _:b606264208 . _:b606264091 "4"^^ . _:b606264376 . . _:b606264377 . . _:b606264365 . _:b606264378 . _:b606264379 . _:b606264090 "4"^^ . _:b606264388 . _:b606264389 . _:b606264390 . _:b606264364 . _:b606264357 . _:b606264391 . _:b606264101 "4"^^ . _:b606264384 . _:b606264385 . _:b606264179 . _:b606264386 . _:b606264408 . _:b606264367 . _:b606264387 . _:b606264100 "4"^^ . _:b606264396 . . _:b606264397 . _:b606264398 . _:b606264366 . _:b606264399 . _:b51921549 "Recently, several newly developed xenotransplantation models have been reported for improved engraftment of human hematopoietic cells including modified NSG strains with overexpression or knock-in of human cytokines 2,4,5,>>45<<,46, co-transplantation of hematopoietic cells with mesenchymal stromal cells 13, and transplantation of engineered bio-scaffolds designed to resemble human BM niches 47." . _:b606264103 "4"^^ . _:b606264392 . _:b606264010 . _:b606264393 . _:b606264394 . _:b606264361 . _:b606264395 . _:b606264102 "4"^^ . _:b606264404 . _:b606264405 . _:b606264406 . _:b606264360 . _:b606264407 . _:b606264097 "4"^^ . _:b606264400 . . _:b606264401 . _:b606264374 . _:b606264402 . _:b606264363 . _:b606264403 . _:b606264096 "4"^^ . _:b606264412 . _:b606264413 . _:b606264414 . _:b606264362 . _:b606264415 . _:b606264099 "4"^^ . _:b606264408 . . _:b606264409 . _:b606264073 . _:b606264016 . _:b606264373 . _:b606264325 . _:b606264410 . _:b606264050 . _:b606264411 . . _:b606264098 "4"^^ . _:b606264218 . _:b606264420 . _:b606264421 . _:b606264169 . _:b606264422 . _:b606264372 . _:b606264109 "3"^^ . _:b606264423 . _:b606264416 . _:b606264352 . _:b606264334 . _:b606264022 . _:b606264417 . _:b606264418 . _:b606264375 . _:b606264108 "3"^^ . _:b606264320 . _:b606264419 . . _:b606264428 . _:b606264429 . . _:b606264374 . _:b606264430 . _:b606264111 "3"^^ . _:b606264431 . _:b606264424 . _:b606264425 . _:b606264121 . _:b606264369 . _:b606264426 . _:b606264427 . _:b606264110 "3"^^ . _:b606264436 . _:b606264437 . _:b606264438 . _:b606264368 . _:b606264439 . _:b606264105 "3"^^ . _:b606264432 . _:b606264433 . . _:b606264434 . _:b606264371 . _:b606264049 . _:b606264435 . _:b51921560 . _:b606264444 . . _:b606264104 "4"^^ . _:b606264445 . . _:b606264446 . _:b606264370 . _:b606264447 . _:b606264021 . _:b606264107 "3"^^ . _:b606264440 . _:b606264441 . _:b606264151 . _:b606264442 . _:b606264381 . _:b606264443 . _:b606264195 . _:b606264106 "3"^^ . _:b606264380 . _:b606264117 "3"^^ . _:b606264315 . _:b606264101 . _:b606264383 . _:b606264116 "3"^^ . _:b606264288 . _:b606264177 . . _:b51921538 . . _:b606264382 . _:b606264119 "3"^^ . . _:b606264377 . _:b606264118 "3"^^ . _:b606264316 . _:b606264376 . _:b606264113 "3"^^ . _:b51921507 . _:b51921542 "In contrast to a recently published report demonstrating establishment of APL from transduced cord blood-derived CMP, we did not identify PML-RARA fusion events in FACS-purified CMP from primary patient samples >>43<<. How PML-RARA initiates a self-renewal program in human progenitors to cause APL is an important question for further studies of APL pathogenesis." . _:b51921550 "Recently, several newly developed xenotransplantation models have been reported for improved engraftment of human hematopoietic cells including modified NSG strains with overexpression or knock-in of human cytokines 2,4,5,45,>>46<<, co-transplantation of hematopoietic cells with mesenchymal stromal cells 13, and transplantation of engineered bio-scaffolds designed to resemble human BM niches 47." . _:b606264379 . _:b606264112 "3"^^ . _:b606264017 . _:b606264378 . _:b606264115 "3"^^ . . _:b606264045 . . _:b606264389 . _:b606264114 "3"^^ . _:b606264449 . _:b606264388 . _:b606264125 "3"^^ . "PMC0" . _:b606264432 . . _:b606264391 . _:b606264124 "3"^^ . _:b606264346 . . _:b606264390 . _:b606264127 "3"^^ . . . _:b606264385 . _:b606264420 . _:b606264126 "3"^^ . _:b606264384 . _:b606264121 "3"^^ . . _:b606264387 . _:b606264120 "3"^^ . _:b606264386 . _:b606264451 . _:b606264123 "3"^^ . _:b606264397 . _:b606264122 "3"^^ . . . _:b606264396 . _:b606264133 "3"^^ . _:b606264184 . _:b606264399 . _:b606264132 "3"^^ . _:b606264104 . _:b606264201 . . _:b606264398 . . _:b606264135 "3"^^ . . _:b51921559 "Allele-specific JAK2 genotyping assays were performed as described previously.>>53<< Briefly, engrafted human B cells (huCD45+CD19+) and myeloid cells (huCD45+CD33+) were sorted on an Aria II flow cytometer (BD) for analysis." . . _:b606264393 . _:b606264134 "3"^^ . _:b606264392 . _:b606264129 "3"^^ . . _:b606264395 . _:b606264128 "3"^^ . _:b51921555 "in four-layered cell factories (CF-4, Thermo Fisher, Nunc, Pittsburg, PA) with half-weekly medium changes until cells reached confluence. At the end of passage 1 (p1), BM-MSC were profiled for expression of consensus MSC surface markers.>>51<< Briefly, trypsinized cells were blocked with sheep serum (10% v/v) and thereafter stained with following monoclonal antibodies for 30 min, 4\u00B0C in the dark:" . _:b606264394 . _:b606264131 "3"^^ . . _:b606264396 . _:b606264303 . _:b606264405 . _:b606264130 "3"^^ . _:b606264404 . _:b51921541 . _:b606264141 "3"^^ . . _:b606264407 . _:b606264140 "3"^^ . _:b606264406 . _:b606264143 "3"^^ . . _:b606264401 . _:b606264142 "3"^^ . . _:b606264400 . _:b606264137 "3"^^ . . . _:b606264403 . _:b606264136 "3"^^ . _:b606264402 . _:b606264139 "3"^^ . _:b606264413 . _:b606264138 "3"^^ . . _:b606264060 . _:b606264412 . _:b606264442 . _:b606264149 "3"^^ . . _:b606264266 . _:b606264415 . _:b606264148 "3"^^ . _:b606264030 . _:b606264026 . . _:b606264414 . _:b606264151 "3"^^ . _:b606264409 . _:b606264150 "3"^^ . _:b606264408 . _:b606264145 "3"^^ . . . _:b606264411 . _:b606264144 "3"^^ . . . . _:b606264410 . _:b606264147 "3"^^ . . _:b606264421 . _:b606264146 "3"^^ . _:b606264420 . _:b606264113 . _:b606264157 "3"^^ . _:b606264013 . . _:b606264423 . _:b606264012 . _:b606264156 "3"^^ . . _:b606264422 . _:b606264015 . _:b51921525 . _:b606264159 "3"^^ . . _:b606264286 . _:b51921513 . _:b606264417 . _:b606264014 . _:b606264158 "3"^^ . . _:b606264183 . _:b606264416 . _:b606264009 . _:b606264153 "3"^^ . _:b51921515 . . _:b606264419 . _:b606264355 . . . _:b606264281 . _:b606264152 "3"^^ . . _:b606264418 . _:b51921537 "APL, the nature of the hematopoietic cell that is the target of the disease-causing chromosomal translocation is a matter of debate24 with in vitro studies implicating immature compartments able to produce myeloid and erythroid output >>36<<, and alternative reports supporting APL arises from committed myeloid progenitors." . _:b606264011 . _:b606264155 "3"^^ . . . _:b606264429 . _:b606264010 . _:b606264154 "3"^^ . _:b51921528 "The successful engraftment of primary human MF cells provided the opportunity to investigate leukemia-initiating cells in this disease, which have long been hypothesized to reside in the HSC compartment >>32<<. We first fractionated CD34+ cells from MF samples into CD34+CD38+ and CD34+CD38lo/- subfractions and transplanted each directly into multiple humanized ossicles (n = 4 \u2013 7 per sample)." . _:b606264428 . _:b606264021 . _:b606264165 "3"^^ . . _:b606264431 . _:b606264020 . _:b606264164 "3"^^ . . . _:b606264041 . _:b606264430 . _:b606264023 . _:b606264167 "3"^^ . _:b606264399 . . _:b606264425 . _:b606264022 . _:b606264166 "3"^^ . _:b606264424 . _:b51921508 . _:b606264017 . _:b606264434 . _:b606264161 "3"^^ . . _:b606264427 . _:b51921509 . _:b606264016 . _:b606264160 "3"^^ . . _:b51921510 . _:b606264426 . _:b606264019 . _:b606264163 "3"^^ . _:b606264437 . _:b51921511 . _:b606264018 . _:b606264162 "3"^^ . . . _:b606264436 . _:b51921504 . _:b606264173 "3"^^ . _:b606264029 . . _:b51921505 . _:b606264439 . _:b606264028 . _:b606264172 "3"^^ . _:b606264310 . _:b606264438 . _:b51921506 . . _:b606264175 "3"^^ . _:b606264031 . _:b606264433 . _:b51921507 . _:b606264030 . _:b606264174 "3"^^ . . _:b606264432 . _:b51921516 . _:b606264025 . _:b606264169 "3"^^ . . _:b606264435 . _:b51921540 . _:b51921517 . . _:b51921522 "Original studies using limiting dilution xenograft assays suggested that leukemia-initiating cells (LIC) in AML were rare, comprising 1 : 10,000 or fewer cells 22,>>23<<. In order to determine if transplantation of AML cells into a humanized microenvironment can facilitate increased engraftment and frequency of LIC, we conducted limiting dilution assays with two AML samples using both direct intraossicle" . _:b606264168 "3"^^ . _:b606264024 . . _:b606264434 . _:b51921518 . _:b606264027 . _:b606264171 "3"^^ . _:b606264445 . _:b51921519 . _:b606264026 . _:b606264170 "3"^^ . . _:b606264444 . _:b51921512 . _:b606264037 . _:b606264181 "3"^^ . . _:b606264219 . _:b606264447 . _:b51921513 . _:b606264036 . _:b606264180 "3"^^ . _:b606264348 . _:b606264425 . . _:b51921525 "The successful engraftment of primary human APL cells provided the opportunity to investigate leukemia-initiating cells (LIC) in this disease, which has been a point of controversy in the field.>>24<< First, we isolated a CD34+CD38-CD45RA- fraction that normally contains HSC and MPP from primary APL samples and compared engraftment of these cells to bulk APL cells in humanized ossicle niches by direct intraossicle injection." . _:b606264446 . _:b606264183 "2"^^ . _:b51921514 . _:b606264039 . _:b606264441 . _:b51921562 . _:b51921515 . _:b606264038 . _:b606264182 "3"^^ . _:b606264059 . _:b606264304 . _:b606264440 . _:b51921524 . _:b606264033 . _:b606264177 "3"^^ . . . _:b606264443 . _:b51921525 . _:b606264032 . _:b606264176 "3"^^ . . _:b606264333 . _:b51921526 . _:b606264442 . . _:b606264179 "3"^^ . _:b606264035 . _:b51921527 . _:b606264034 . _:b606264178 "3"^^ . _:b606264251 . . _:b606264189 "2"^^ . _:b51921520 . _:b606264045 . _:b606264223 . _:b606264188 "2"^^ . _:b51921521 . _:b606264342 . _:b606264044 . _:b606264351 . _:b606264191 "2"^^ . _:b51921522 . _:b606264047 . _:b606264190 "2"^^ . _:b51921523 . _:b606264449 . _:b606264046 . . _:b51921536 "In APL, the nature of the hematopoietic cell that is the target of the disease-causing chromosomal translocation is a matter of debate>>24<< with in vitro studies implicating immature compartments able to produce myeloid and erythroid output 36, and alternative reports supporting APL arises from committed myeloid progenitors." . . _:b606264185 "2"^^ . _:b51921532 . _:b606264448 . _:b606264041 . _:b606264411 . _:b606264184 "2"^^ . _:b51921537 . _:b51921533 . _:b606264451 . _:b606264040 . . _:b606264187 "2"^^ . _:b51921534 . _:b606264450 . _:b606264043 . . . _:b51921535 . _:b606264186 "2"^^ . . _:b606264042 . . _:b51921517 . _:b606264191 . _:b606264197 "2"^^ . _:b51921528 . _:b606264053 . _:b606264284 . _:b606264196 "2"^^ . _:b51921529 . . _:b606264052 . . _:b606264199 "2"^^ . _:b51921530 . _:b606264055 . _:b606264198 "2"^^ . _:b51921531 . _:b606264324 . . _:b606264054 . _:b606264252 . _:b606264061 . . _:b606264193 "2"^^ . _:b51921540 . _:b606264049 . _:b606264278 . _:b606264306 . . _:b51921541 . _:b606264192 "2"^^ . _:b606264048 . _:b606264029 . _:b51921542 . _:b606264195 "2"^^ . _:b606264192 . _:b606264051 . _:b606264194 "2"^^ . _:b51921543 . _:b606264114 . _:b606264050 . _:b606264098 . . _:b606264097 . _:b606264205 "2"^^ . _:b51921536 . _:b606264061 . _:b51921524 "Transplantation of human acute promyelocytic leukemia (APL) into immunocompromised mice has so far been of limited success with low to no detectable engraftment 9,>>23<<. Since humanized ossicle niches show superior engraftment of non-APL AML samples, we investigated the engraftment of primary human APL cells by direct intraossicle injection." . _:b606264204 "2"^^ . _:b51921537 . _:b606264060 . _:b606264415 . _:b606264347 . _:b606264207 "2"^^ . _:b51921538 . _:b606264063 . . . _:b606264206 "2"^^ . _:b51921539 . _:b606264062 . . . _:b606264031 . _:b51921548 . _:b606264337 . _:b606264201 "2"^^ . _:b606264057 . _:b606264367 . . . _:b51921549 . _:b606264385 . _:b606264200 "2"^^ . _:b606264056 . . _:b606264203 "2"^^ . _:b51921550 . _:b606264293 . _:b606264059 . _:b606264202 "2"^^ . _:b51921551 . . _:b606264381 . _:b606264058 . _:b606264213 "2"^^ . _:b51921544 . . . . _:b606264212 "2"^^ . _:b51921545 . . _:b606264215 "2"^^ . _:b51921546 . _:b606264401 . . . _:b606264214 "2"^^ . _:b51921547 . _:b606264243 . _:b606264209 "2"^^ . _:b51921556 . . _:b606264294 . . . . _:b51921557 . _:b606264208 "2"^^ . . _:b606264289 . _:b606264105 . _:b606264417 . _:b606264211 "2"^^ . _:b51921558 . _:b606264210 "2"^^ . _:b51921559 . _:b51921532 "Original xenotransplantation studies suggested AML LIC/LSC were rare cells, on the order of 1 : 10,000 \u2013 100,000 enriched in the CD34+CD38- fraction of leukemic blasts 22,>>23<<. More recent studies suggested that LIC/LSC are more frequent and can be found in additional immunophenotypically defined subpopulations 7,34,35. Here, we provide evidence that with a humanized niche, the frequency of LIC is substantially" . _:b606264221 "2"^^ . _:b606264446 . _:b51921552 . . _:b51921553 . _:b606264220 "2"^^ . _:b606264126 . _:b606264270 . _:b606264223 "2"^^ . _:b51921554 . _:b51921551 "strains with overexpression or knock-in of human cytokines 2,4,5,45,46, co-transplantation of hematopoietic cells with mesenchymal stromal cells 13, and transplantation of engineered bio-scaffolds designed to resemble human BM niches >>47<<. While each of these approaches has advantages compared to conventional NSG mouse models, they have not demonstrated the rapid and high leukemic engraftment observed with our humanized ossicle niche model, where direct intraossicle" . . _:b606264222 "2"^^ . _:b51921555 . . _:b606264217 "2"^^ . _:b606264272 . _:b606264216 "2"^^ . _:b51921531 . _:b606264219 "2"^^ . _:b606264387 . _:b606264218 "2"^^ . . _:b606264229 "2"^^ . _:b51921560 . _:b606264228 "2"^^ . _:b51921561 . _:b606264231 "2"^^ . _:b51921562 . . _:b606264068 . _:b606264230 "2"^^ . _:b606264225 "2"^^ . _:b606264224 "2"^^ . _:b606264205 . _:b606264227 "2"^^ . _:b606264080 . _:b606264226 "2"^^ . _:b606264424 . _:b51921521 . . _:b606264312 . . _:b51921515 "We sought to establish a xenotransplantation system for human normal and malignant hematopoietic cells through the generation of in vivo humanized BM niches in NSG mice >>17<< (Fig. 1a and see Supplementary Fig. 1a-f for a detailed protocol for humanized ossicle formation)." . _:b606264237 "2"^^ . _:b606264247 . _:b606264236 "2"^^ . _:b606264395 . . . _:b51921546 "Recently, several newly developed xenotransplantation models have been reported for improved engraftment of human hematopoietic cells including modified NSG strains with overexpression or knock-in of human cytokines >>2<<,4,5,45,46, co-transplantation of hematopoietic cells with mesenchymal stromal cells 13, and transplantation of engineered bio-scaffolds designed to resemble human BM niches 47." . . _:b606264239 "2"^^ . _:b606264238 "2"^^ . . _:b51921535 . _:b606264233 "2"^^ . . _:b606264232 "2"^^ . _:b51921526 . _:b606264235 "2"^^ . _:b51921526 . . . _:b606264234 "2"^^ . . _:b51921508 . _:b606264245 "2"^^ . _:b606264145 . _:b606264244 "2"^^ . . _:b606264247 "2"^^ . _:b606264318 . _:b606264206 . _:b606264246 "2"^^ . _:b606264241 "2"^^ . _:b606264240 "2"^^ . _:b606264290 . . _:b606264118 . . _:b606264243 "2"^^ . _:b51921524 . _:b606264242 "2"^^ . _:b606264253 "2"^^ . _:b606264269 . . _:b606264252 "2"^^ . . _:b606264255 "2"^^ . _:b606264375 . . . _:b606264254 "2"^^ . . _:b606264249 "2"^^ . . _:b51921522 . _:b606264248 "2"^^ . _:b606264251 "2"^^ . _:b606264349 . . . _:b51921532 . _:b606264250 "2"^^ . _:b606264261 "2"^^ . _:b606264074 . . _:b606264260 "2"^^ . . _:b51921535 "More recent studies suggested that LIC/LSC are more frequent and can be found in additional immunophenotypically defined subpopulations 7,34,>>35<<. Here, we provide evidence that with a humanized niche, the frequency of LIC is substantially higher than that determined in the NSG mouse BM with the same sample, suggesting that AML LIC frequency is critically dependent on leukemia" . _:b606264263 "2"^^ . . . _:b606264262 "2"^^ . . . _:b606264257 "2"^^ . _:b606264153 . _:b606264256 "2"^^ . _:b51921536 . _:b606264253 . _:b606264259 "2"^^ . _:b606264258 "2"^^ . _:b606264258 . _:b606264140 . _:b606264269 "2"^^ . _:b606264216 . _:b606264268 "2"^^ . _:b51921521 "Original studies using limiting dilution xenograft assays suggested that leukemia-initiating cells (LIC) in AML were rare, comprising 1 : 10,000 or fewer cells >>22<<,23. In order to determine if transplantation of AML cells into a humanized microenvironment can facilitate increased engraftment and frequency of LIC, we conducted limiting dilution assays with two AML samples using both direct" . . . _:b606264271 "2"^^ . . _:b606264270 "2"^^ . _:b606264185 . _:b51921514 "results" . . _:b606264158 . _:b606264265 "2"^^ . _:b606264264 "2"^^ . . _:b606264267 "2"^^ . . _:b606264076 . _:b606264295 . . . _:b606264266 "2"^^ . . _:b606264277 "2"^^ . . _:b606264065 . _:b606264276 "2"^^ . . . . _:b606264279 "2"^^ . _:b606264299 . _:b606264278 "2"^^ . _:b606264368 . . . _:b606264273 "2"^^ . _:b606264079 . . _:b606264272 "2"^^ . _:b606264275 "2"^^ . _:b51921554 "Human BM samples were obtained according to Medical University of Graz Ethikkommission (Institutional Review Board-approved protocol, MUG Graz IRB no. 19-252). BM-MSC were isolated and expanded as previously described.17,49,>>50<< Age of healthy BM donors ranged from 21 \u2013 45 years. Briefly, BM-MNCs were washed out from BM collection filters by flushing the filters with pre-warmed PBS." . _:b606264274 "2"^^ . . _:b606264285 "2"^^ . . _:b606264284 "2"^^ . _:b606264412 . _:b606264287 "2"^^ . . . _:b606264286 "2"^^ . _:b606264281 "2"^^ . _:b51921528 . _:b606264280 "2"^^ . . _:b606264283 "2"^^ . . _:b606264282 "2"^^ . . _:b51921542 . _:b606264149 . _:b606264293 "2"^^ . . . _:b606264292 "2"^^ . _:b606264295 "2"^^ . _:b606264262 . _:b606264294 "2"^^ . _:b606264419 . _:b606264289 "2"^^ . . _:b606264165 . _:b606264288 "2"^^ . . _:b51921510 "In these niches, HSC reside in close contact and bidirectional interaction with a complex network of cells including MSC, osteoblasts, adipocytes, vascular endothelial cells, and Schwann cells >>14<<. These niches not only provide sanctuary sites for HSC, but are also co-opted by leukemia cells in hematopoietic malignancies and can support LSC survival 15,16." . _:b606264291 "2"^^ . . . _:b606264290 "2"^^ . . . _:b606264301 "2"^^ . _:b606264302 . _:b606264055 . _:b606264246 . _:b606264300 "2"^^ . _:b606264217 . _:b606264303 "2"^^ . _:b606264232 . _:b606264302 "2"^^ . _:b606264297 "2"^^ . . _:b606264120 . . _:b606264296 "2"^^ . . . _:b606264299 "2"^^ . _:b606264422 . _:b606264298 "2"^^ . _:b606264146 . _:b606264382 . _:b606264413 . _:b606264309 "2"^^ . . _:b606264308 "2"^^ . _:b606264225 . _:b606264238 . _:b606264311 "2"^^ . . _:b606264058 . _:b606264310 "2"^^ . . . _:b606264305 "2"^^ . _:b606264304 "2"^^ . _:b606264307 "2"^^ . . _:b606264203 . . _:b606264069 . _:b606264306 "2"^^ . . _:b606264068 . _:b606264384 . _:b606264317 "2"^^ . . _:b606264071 . _:b606264316 "2"^^ . _:b606264448 . _:b51921548 "Recently, several newly developed xenotransplantation models have been reported for improved engraftment of human hematopoietic cells including modified NSG strains with overexpression or knock-in of human cytokines 2,4,>>5<<,45,46, co-transplantation of hematopoietic cells with mesenchymal stromal cells 13, and transplantation of engineered bio-scaffolds designed to resemble human BM niches 47." . _:b606264070 . _:b606264319 "2"^^ . . _:b606264376 . _:b606264065 . _:b606264318 "2"^^ . . _:b606264313 "2"^^ . _:b606264064 . _:b606264067 . _:b606264312 "2"^^ . _:b606264311 . _:b606264066 . _:b606264315 "2"^^ . _:b606264077 . _:b606264364 . _:b606264314 "2"^^ . . . _:b606264110 . _:b606264076 . _:b606264137 . . _:b606264079 . _:b606264129 . _:b606264078 . _:b606264321 . _:b606264073 . . _:b606264072 . _:b606264075 . _:b606264139 . _:b606264341 . _:b606264074 . . . _:b606264085 . . _:b51921543 "Similar to APL, our humanized ossicle model facilitates robust engraftment of primary human MF cells, which has only been of limited success with peripheral blood derived cells with conventional NSG models.>>31<<,44 Unlike studies of AML, where disease-initiating cells arise from progenitors 35, we show here that myelofibrosis-initiating cells reside in the HSC compartment, as transplantation of these cells, but not MPP or CMP, results in" . _:b606264084 . . . _:b606264087 . _:b606264086 . . _:b606264081 . _:b606264080 . _:b51921533 "More recent studies suggested that LIC/LSC are more frequent and can be found in additional immunophenotypically defined subpopulations >>7<<,34,35. Here, we provide evidence that with a humanized niche, the frequency of LIC is substantially higher than that determined in the NSG mouse BM with the same sample, suggesting that AML LIC frequency is critically dependent on" . . _:b606264237 . _:b606264372 . _:b606264116 . _:b606264083 . . _:b606264082 . _:b606264181 . _:b606264256 . . . _:b606264093 . . _:b606264092 . _:b606264285 . _:b606264095 . . . . . _:b606264094 . . _:b606264089 . _:b606264084 . _:b606264088 . . _:b606264091 . _:b606264131 . _:b51921546 . . _:b606264397 . _:b606264090 . . _:b606264101 . _:b606264345 . _:b606264445 . . _:b606264100 . _:b606264103 . . _:b606264102 . _:b606264097 . . . _:b606264096 . . _:b606264099 . _:b606264406 . . _:b606264098 . . _:b606264109 . . _:b606264369 . _:b606264331 . _:b606264108 . _:b51921538 . _:b606264111 . _:b606264110 . _:b606264090 . _:b606264105 . _:b606264190 . _:b606264104 . _:b51921519 "Note that in the non-transplanted ossicles, human leukemia cells were almost exclusively localized to the endosteal paratrabecular regions previously demonstrated to be preferred niches for both normal HSC and AML LSC 15,>>20<<." . . . _:b606264107 . _:b606264280 . _:b606264106 . _:b606264211 . _:b606264117 . _:b606264127 . _:b606264116 . _:b606264265 . _:b606264119 . _:b606264199 . _:b606264118 . _:b606264230 . _:b606264093 . _:b606264371 . _:b606264113 . _:b606264095 . _:b606264112 . _:b606264291 . _:b51921508 "However, a large proportion of AML patient samples, in particular less aggressive subtypes such as core binding factor mutants and acute promyelocytic leukemia (APL), fail to engraft or do so at low levels that do not mimic human disease >>7<<-9. Additionally, many other hematopoietic neoplasms do not engraft in the currently available mouse strains, as transplantation of MDS, MPN, or multiple myeloma has met with very limited success 10-12, although in myelodysplastic syndrome" . _:b606264115 . _:b606264161 . _:b606264114 . _:b606264125 . _:b606264282 . . . . _:b606264069 . _:b606264124 . _:b606264127 . . _:b606264383 . _:b606264336 . . . _:b606264126 . _:b51921550 . _:b606264121 . _:b51921505 . . . . _:b606264120 . _:b606264175 . _:b606264123 . _:b51921526 "Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) commonly associated with JAK2V617F and calreticulin (CALR) mutations >>25<<-28 that has shown only limited engraftment with transplantation of large numbers of patient-derived CD34+ cells in conventional xenograft models 29-31." . _:b606264330 . _:b606264122 . . _:b51921519 . . . _:b606264133 . _:b606264132 . _:b606264135 . _:b606264134 . _:b606264200 . _:b606264129 . _:b606264128 . . _:b606264167 . . . _:b606264329 . _:b606264131 . _:b606264087 . _:b51921506 . _:b606264130 . . _:b606264250 . _:b606264141 . . _:b606264140 . _:b606264410 . _:b606264143 . _:b606264142 . _:b606264012 . _:b606264228 . _:b606264137 . _:b51921507 "These mouse strains have been used extensively for the engraftment of human hematopoietic malignancies, particularly acute myeloid leukemia (AML) >>6<<. However, a large proportion of AML patient samples, in particular less aggressive subtypes such as core binding factor mutants and acute promyelocytic leukemia (APL), fail to engraft or do so at low levels that do not mimic human disease" . _:b606264178 . _:b606264171 . _:b606264136 . _:b606264188 . _:b606264130 . _:b606264139 . _:b606264138 . _:b606264338 . _:b606264308 . _:b606264365 . _:b606264149 . _:b606264248 . _:b606264344 . _:b51921516 . . _:b606264148 . _:b606264222 . _:b606264151 . . _:b606264150 . _:b606264145 . _:b51921548 . _:b606264144 . . _:b606264147 . _:b51921538 . _:b606264146 . . . _:b606264157 . . _:b606264263 . . _:b606264156 . _:b606264236 . _:b606264159 . _:b606264158 . _:b606264153 . _:b606264421 . _:b606264391 . _:b51921513 "that immature mesenchymal stromal cells from human BM (BM-MSC) can recreate a functional hematopoietic microenvironment in NSG mice upon transplantation into ectopic sites through endochondral ossification to form a humanized ossicle >>17<<. We speculated that these ossicles contain a humanized microenvironment with the proper supply of human niche factors to facilitate superior engraftment and growth of normal and malignant human hematopoietic cells." . _:b606264124 . _:b606264152 . . _:b606264231 . _:b51921549 . _:b606264155 . . _:b606264154 . _:b606264276 . _:b606264165 . _:b606264264 . _:b606264164 . _:b606264167 . . _:b606264166 . _:b606264161 . . _:b606264160 . _:b606264163 . _:b606264173 . _:b606264162 . _:b606264322 . _:b606264173 . _:b606264354 . . _:b51921527 . _:b606264292 . _:b606264172 . . _:b606264175 . _:b606264111 . _:b606264363 . _:b606264174 . . _:b606264169 . _:b606264403 . _:b606264066 . . _:b606264168 . _:b606264020 . _:b606264086 . _:b606264171 . _:b606264170 . _:b606264176 . . _:b606264181 . _:b51921560 "The native samples (prior to xenotransplantation) were genotyped by whole exome sequencing (SeqCap EZ Exome SR kit v3.0, Roche/Nimblegen) as previously described >>54<<. All kits were used following manufacturer's instructions (Roche/Nimblegen). Illumina HiSeq 2000, HiSeq 2500, or NextSeq 500 instruments were used for sequencing." . _:b606264180 . _:b606264214 . _:b606264183 . _:b606264067 . _:b606264182 . . _:b606264177 . _:b606264176 . . . _:b606264179 . _:b606264182 . . . . . _:b51921529 "B into HSC and MPP>>33<<, and tested their engraftment potential through direct intraossicle injection." . _:b606264178 . _:b606264393 . _:b606264032 . _:b606264189 . _:b606264011 . _:b606264188 . . . _:b606264191 . _:b606264323 . _:b606264102 . _:b606264190 . . . _:b606264185 . . _:b606264443 . . _:b606264184 . _:b606264187 . . . _:b606264172 . _:b606264186 . . _:b606264197 . _:b606264440 . _:b606264196 . . _:b606264199 . _:b606264198 . . _:b606264296 . _:b606264193 . . _:b606264155 . _:b606264192 . . _:b51921520 . _:b606264227 . _:b606264195 . _:b606264194 . _:b606264077 . _:b606264196 . _:b606264377 . _:b606264388 . _:b606264205 . . . _:b606264144 . . _:b606264204 . . _:b606264207 . . _:b606264012 . _:b606264013 . _:b606264409 . _:b606264014 . _:b606264319 . _:b606264206 . . _:b606264015 . _:b606264447 . _:b606264343 . _:b606264198 . _:b606264423 . _:b606264009 . _:b51921531 "Original xenotransplantation studies suggested AML LIC/LSC were rare cells, on the order of 1 : 10,000 \u2013 100,000 enriched in the CD34+CD38- fraction of leukemic blasts >>22<<,23. More recent studies suggested that LIC/LSC are more frequent and can be found in additional immunophenotypically defined subpopulations 7,34,35. Here, we provide evidence that with a humanized niche, the frequency of LIC is" . _:b606264043 . _:b606264010 . _:b606264201 . _:b606264011 . _:b606264020 . _:b606264166 . . _:b606264021 . . _:b606264022 . _:b606264200 . _:b606264023 . _:b606264016 . _:b606264017 . _:b606264018 . _:b606264203 . _:b606264019 . _:b606264028 . . _:b606264029 . . _:b606264202 . _:b606264030 . . _:b606264031 . _:b606264024 . _:b606264300 . _:b606264025 . _:b606264026 . _:b606264213 . _:b606264027 . _:b606264036 . . _:b606264037 . . . _:b606264212 . _:b606264038 . _:b606264039 . _:b606264032 . _:b606264033 . _:b606264034 . _:b606264215 . . _:b606264035 . _:b606264044 . _:b606264045 . _:b606264046 . _:b606264214 . _:b606264047 . . . _:b606264040 . _:b606264041 . _:b606264053 . _:b606264042 . _:b606264209 . . _:b606264043 . _:b606264052 . _:b606264053 . . _:b606264054 . _:b606264208 . . _:b606264055 . . _:b606264048 . _:b606264049 . _:b606264050 . _:b606264211 . _:b606264051 . _:b606264204 . _:b606264060 . _:b606264061 . _:b606264062 . _:b606264210 . _:b606264063 . _:b606264056 . . _:b606264057 . _:b606264058 . _:b606264221 . _:b606264059 . _:b51921540 "These results are consistent with mouse models that drive expression of PML-RARA from a myeloid lineage-restricted promoter resulting in an APL-like disease 40,>>41<<. The absence of PML-RARA translocation in human HSC obtained from APL patient samples is surprising given that PML-RARA on its own induced myeloid commitment rather than self-renewal in hematopoietic progenitors such as GMP 42. In" . _:b606264220 . _:b606264125 . . . _:b606264350 . _:b606264223 . _:b606264362 . _:b606264222 . _:b606264234 . _:b606264217 . _:b606264216 . . . _:b51921509 . _:b606264219 . _:b606264162 . _:b606264218 . . . _:b606264229 . _:b606264360 . _:b606264228 . . _:b606264231 . _:b606264230 . _:b606264225 . _:b606264229 . _:b606264224 . _:b606264227 . _:b51921545 . _:b606264168 . _:b606264226 . _:b606264237 . _:b606264212 . _:b606264236 . _:b606264071 . . _:b606264239 . _:b606264136 . _:b606264238 . _:b606264233 . _:b51921530 "discussion" . _:b606264260 . . . _:b606264232 . . _:b606264235 . . _:b606264234 . _:b51921543 . . _:b606264245 . _:b606264106 . _:b606264028 . _:b51921545 "31,44 Unlike studies of AML, where disease-initiating cells arise from progenitors >>35<<, we show here that myelofibrosis-initiating cells reside in the HSC compartment, as transplantation of these cells, but not MPP or CMP, results in engraftment of JAK2V617F or CALR-mutant cells." . . _:b606264244 . _:b606264247 . _:b606264096 . _:b51921506 . _:b606264246 . _:b606264450 . _:b606264244 . _:b606264241 . _:b606264259 . _:b606264240 . . . _:b606264243 . . _:b51921553 "Human BM samples were obtained according to Medical University of Graz Ethikkommission (Institutional Review Board-approved protocol, MUG Graz IRB no. 19-252). BM-MSC were isolated and expanded as previously described.>>17<<,49,50 Age of healthy BM donors ranged from 21 \u2013 45 years. Briefly, BM-MNCs were washed out from BM collection filters by flushing the filters with pre-warmed PBS." . _:b51921547 "Recently, several newly developed xenotransplantation models have been reported for improved engraftment of human hematopoietic cells including modified NSG strains with overexpression or knock-in of human cytokines 2,>>4<<,5,45,46, co-transplantation of hematopoietic cells with mesenchymal stromal cells 13, and transplantation of engineered bio-scaffolds designed to resemble human BM niches 47." . _:b606264242 . . . . . _:b606264253 . _:b606264196 . _:b51921556 . _:b606264197 . _:b606264198 . _:b606264252 . _:b606264199 . . _:b606264192 . _:b606264193 . . _:b606264194 . _:b606264255 . _:b606264054 . _:b606264195 . _:b606264204 . _:b606264205 . . _:b606264206 . _:b606264254 . _:b606264207 . . _:b606264200 . _:b606264201 . _:b606264202 . _:b606264249 . _:b606264107 . _:b606264203 . _:b606264283 . _:b606264212 . . _:b606264213 . _:b606264214 . _:b606264248 . _:b606264220 . _:b606264215 . _:b606264208 . _:b606264209 . _:b606264210 . _:b606264251 . _:b606264211 . _:b606264220 . _:b606264221 . . _:b606264222 . _:b606264250 . _:b606264042 . _:b606264223 . _:b606264216 . _:b606264217 . _:b606264240 . _:b606264218 . _:b606264261 . _:b606264219 . _:b606264228 . _:b606264229 . _:b606264230 . _:b606264260 . . _:b606264231 . . _:b606264340 . _:b606264224 . _:b606264225 . _:b606264226 . _:b606264263 . _:b606264227 . _:b606264236 . _:b606264271 . . _:b606264237 . _:b606264238 . _:b606264262 . . . _:b606264239 . _:b606264232 . . . _:b606264233 . _:b606264234 . _:b606264257 . _:b51921504 _:b51921508 . _:b606264235 . _:b51921504 _:b51921509 . _:b606264244 . _:b51921504 _:b51921510 . _:b606264245 . _:b51921504 _:b51921511 . _:b606264246 . _:b51921526 . _:b606264256 . _:b606264247 . _:b51921504 _:b51921505 . _:b606264240 . _:b51921504 _:b51921506 . _:b606264241 . _:b51921504 _:b51921507 . _:b606264242 . _:b606264430 . _:b606264259 . _:b606264243 . _:b606264252 . _:b606264253 . . _:b606264254 . _:b606264258 . _:b51921504 _:b51921512 . . _:b606264255 . _:b51921504 _:b51921513 . _:b606264248 . _:b606264249 . . _:b606264250 . _:b606264269 . _:b606264251 . _:b606264202 . _:b606264260 . _:b606264261 . _:b606264262 . _:b606264268 . . _:b606264263 . _:b606264256 . _:b606264257 . _:b606264258 . _:b606264271 . . _:b606264259 . _:b606264268 . _:b51921557 "subcutaneous injections of human parathyroid hormone (PTH [1-34]; R&D Systems, Minneapolis, MN; 40 \u03BCg/kg body weight; dorsal neck fold) for 28 consecutive days to further promote bone marrow niche formation, as previously described.18,>>19<< Eight \u2013 10 weeks post BM-MSC application the site of injection was shaved and transplants were evaluated for bone and marrow formation by palpation and by visual inspection (development of a purple hue is indicative of bone marrow niche" . . _:b606264269 . _:b606264270 . _:b606264270 . _:b606264271 . _:b606264264 . . _:b606264328 . _:b606264241 . _:b606264265 . _:b606264266 . _:b606264265 . _:b51921514 _:b51921516 . . _:b51921514 _:b51921517 . _:b606264267 . _:b606264276 . _:b51921514 _:b51921518 . _:b606264099 . _:b51921514 _:b51921519 . _:b606264277 . _:b606264278 . _:b606264264 . _:b606264163 . . _:b606264279 . _:b606264272 . _:b606264273 . _:b606264429 . _:b51921514 _:b51921515 . . _:b606264267 . _:b606264274 . _:b606264275 . _:b606264284 . _:b606264081 . _:b606264285 . _:b606264035 . _:b606264115 . _:b606264286 . _:b606264266 . _:b606264287 . _:b606264280 . _:b606264370 . . _:b606264281 . _:b606264282 . _:b606264277 . _:b606264283 . _:b51921517 "left panel). Daily administration of an anabolic dose of human parathyroid hormone (40 \u03BCg/kg) for 28 days after BM-MSC transplantation resulted in a significant increase in the size of the humanized ossicles (Supplementary Fig. 1f).18,>>19<< In order to confirm the human origin of ossicle bone and stromal niche elements, BM-MSC were transduced with lentivirus to stably express green fluorescent protein (GFP)." . . _:b606264292 . _:b606264293 . _:b606264353 . _:b606264294 . _:b606264276 . _:b51921514 _:b51921528 . _:b606264295 . _:b51921514 _:b51921529 . _:b606264288 . _:b51921510 . _:b606264289 . _:b606264290 . _:b606264279 . _:b51921514 _:b51921524 . _:b606264291 . _:b51921514 _:b51921525 . _:b606264300 . _:b51921514 _:b51921526 . _:b606264036 . _:b606264301 . _:b51921514 _:b51921527 . _:b606264302 . _:b606264278 . _:b51921514 _:b51921520 . _:b606264303 . _:b51921514 _:b51921521 . _:b606264051 . _:b606264296 . _:b51921514 _:b51921522 . . _:b606264297 . _:b606264221 . _:b51921514 _:b51921523 . _:b606264298 . _:b606264273 . _:b606264299 . _:b606264308 . _:b606264309 . _:b51921562 "The absolute number of total mouse BM cells was extrapolated as previously described.>>55<< To calculate total human leukemia cells engrafted within the total humanized ossicle-niche space as compared to the mouse BM, the percentage of human CD45+CD33+ (determined by flow-cytometry) was multiplied with the maximal number of" . _:b606264310 . _:b606264272 . . . _:b606264311 . _:b606264304 . _:b606264402 . _:b606264305 . _:b51921506 . _:b606264152 . _:b606264306 . _:b606264275 . _:b606264307 . _:b606264316 . _:b606264325 "2"^^ . _:b606264317 . _:b606264318 . _:b606264274 . _:b606264319 . _:b606264312 . _:b606264324 "2"^^ . _:b606264313 . . _:b606264285 . _:b606264314 . _:b606264315 . _:b606264068 . _:b606264327 "2"^^ . _:b606264014 . _:b606264069 . _:b606264427 . . _:b606264284 . _:b606264070 . . . _:b606264071 . _:b606264064 . _:b606264326 "2"^^ . _:b606264065 . _:b606264066 . _:b606264287 . _:b606264067 . _:b606264117 . _:b606264076 . _:b606264321 "2"^^ . _:b606264077 . _:b606264078 . _:b606264286 . _:b606264079 . . _:b606264072 . _:b606264320 "2"^^ . . _:b606264073 . _:b606264301 . _:b606264074 . _:b606264281 . _:b606264075 . _:b606264084 . _:b606264323 "2"^^ . _:b606264082 . _:b606264085 . _:b606264378 . _:b606264361 . _:b606264086 . _:b606264280 . _:b606264087 . _:b606264080 . _:b606264322 "2"^^ . _:b606264081 . _:b606264082 . _:b606264283 . _:b606264083 . _:b606264092 . _:b51921558 . _:b606264333 "2"^^ . _:b606264093 . _:b606264317 . _:b606264094 . _:b606264282 . _:b606264095 . . _:b606264088 . _:b606264332 "2"^^ . _:b606264089 . _:b606264090 . _:b606264293 . _:b51921530 _:b51921532 . _:b606264091 . _:b51921530 _:b51921533 . . _:b606264438 . _:b606264335 "2"^^ . _:b51921530 _:b51921534 . _:b606264100 . _:b606264101 . _:b51921530 _:b51921535 . . _:b606264292 . _:b606264102 . _:b606264103 . . _:b606264096 . _:b51921559 . _:b606264334 "2"^^ . . _:b606264097 . _:b51921530 _:b51921531 . _:b606264098 . _:b606264398 . _:b606264295 . _:b606264099 . _:b606264108 . _:b606264329 "2"^^ . _:b606264109 . _:b606264314 . _:b606264110 . _:b606264294 . _:b606264111 . _:b606264104 . _:b606264328 "2"^^ . _:b606264105 . _:b606264106 . _:b606264332 . _:b606264289 . _:b51921530 _:b51921548 . _:b606264107 . _:b51921551 . _:b51921530 _:b51921549 . _:b606264047 . _:b606264331 "2"^^ . . _:b51921530 _:b51921550 . _:b606264116 . . _:b606264117 . _:b51921530 _:b51921551 . _:b606264118 . _:b606264288 . _:b51921530 _:b51921544 . _:b606264119 . _:b51921530 _:b51921545 . . _:b606264112 . _:b606264400 . _:b606264330 "2"^^ . _:b51921530 _:b51921546 . . _:b606264113 . _:b51921530 _:b51921547 . _:b606264114 . _:b606264291 . _:b51921530 _:b51921540 . . _:b51921530 _:b51921541 . _:b606264115 . _:b606264124 . _:b606264341 "2"^^ . _:b51921530 _:b51921542 . _:b606264125 . _:b51921530 _:b51921543 . _:b606264126 . _:b51921534 . _:b606264290 . _:b51921530 _:b51921536 . _:b606264127 . _:b51921530 _:b51921537 . _:b606264120 . _:b606264340 "2"^^ . _:b606264268 . _:b606264226 . _:b51921530 _:b51921538 . _:b606264121 . _:b51921530 _:b51921539 . _:b606264122 . _:b606264301 . . _:b606264123 . _:b606264132 . _:b606264435 . _:b606264343 "2"^^ . _:b606264133 . . _:b606264134 . _:b51921518 "Note that in the non-transplanted ossicles, human leukemia cells were almost exclusively localized to the endosteal paratrabecular regions previously demonstrated to be preferred niches for both normal HSC and AML LSC >>15<<,20." . _:b606264300 . . _:b606264135 . _:b606264128 . _:b606264342 "2"^^ . _:b606264023 . _:b606264129 . . _:b606264130 . _:b606264303 . _:b606264131 . _:b606264140 . _:b606264337 "2"^^ . _:b606264141 . _:b606264142 . _:b51921509 . _:b606264302 . _:b606264048 . _:b606264143 . _:b606264136 . _:b606264336 "2"^^ . _:b606264137 . _:b606264138 . _:b51921556 "subcutaneous injections of human parathyroid hormone (PTH [1-34]; R&D Systems, Minneapolis, MN; 40 \u03BCg/kg body weight; dorsal neck fold) for 28 consecutive days to further promote bone marrow niche formation, as previously described.>>18<<,19 Eight \u2013 10 weeks post BM-MSC application the site of injection was shaved and transplants were evaluated for bone and marrow formation by palpation and by visual inspection (development of a purple hue is indicative of bone marrow" . _:b606264297 . _:b606264139 . _:b606264148 . _:b606264428 . _:b606264339 "2"^^ . _:b606264149 . _:b606264150 . _:b606264296 . . _:b606264151 . _:b606264144 . _:b606264338 "2"^^ . _:b606264159 . _:b606264145 . "10.1038%2Fnm.4103" . _:b606264146 . _:b606264299 . _:b606264147 . _:b606264156 . _:b606264349 "2"^^ . _:b606264157 . _:b606264158 . _:b606264298 . _:b606264159 . _:b606264152 . _:b606264348 "2"^^ . _:b606264153 . _:b51921520 "recapitulate the clonal heterogeneity of patient samples, resulting in engrafting subclones that may be distinct from the dominant founding or relapse clone, presumably due to selective pressure imparted by the mouse BM microenvironment >>21<<. We investigated the clonal composition of human AML cells engrafted in the humanized ossicle niches through targeted re-sequencing of known leukemia mutations in four of our engrafted normal karyotype cases." . _:b606264154 . _:b606264309 . _:b606264274 . _:b606264155 . _:b606264164 . _:b606264351 "2"^^ . _:b606264165 . _:b606264166 . _:b606264308 . _:b606264167 . _:b606264160 . _:b606264436 . _:b606264350 "2"^^ . _:b606264161 . . . _:b606264311 . _:b606264162 . _:b606264163 . _:b606264326 . . _:b606264172 . _:b606264345 "2"^^ . . _:b51921561 "Hematoxylin-eosin (H/E) staining and IHC staining were performed using standard protocols >>17<<. For IHC staining, temperature-based antigen retrieval was performed (70 \u00B0C, 160 W, 40 min) followed by a descending alcohol series. Endogenous peroxidases were blocked with hydrogen peroxide (10 min) and nonspecific antibody binding with" . _:b606264173 . _:b606264174 . _:b606264310 . _:b606264175 . _:b606264168 . _:b606264344 "2"^^ . _:b606264169 . _:b606264170 . _:b606264305 . _:b606264171 . . _:b606264180 . _:b606264347 "2"^^ . _:b606264181 . _:b606264170 . . _:b606264182 . _:b606264304 . . _:b51921506 "cell engraftment often remained at low levels leading to the development of further strains with improved xenograft efficiency through overexpression or targeted insertion of human cytokines such as SCF, GM-CSF, IL-3, and TPO >>2<<-5." . _:b606264183 . _:b606264176 . _:b606264346 "2"^^ . _:b606264177 . _:b606264178 . _:b606264307 . _:b606264179 . _:b606264188 . _:b606264357 "2"^^ . _:b606264189 . _:b606264190 . _:b606264306 . _:b606264191 . _:b606264184 . _:b606264356 "2"^^ . _:b606264185 . _:b606264154 . _:b606264186 . _:b606264317 . _:b606264187 . _:b606264359 "2"^^ . . _:b606264316 . . _:b606264128 . _:b606264358 "2"^^ . . _:b606264319 . . _:b606264353 "2"^^ . _:b51921506 . _:b606264318 . . _:b606264352 "2"^^ . . _:b606264313 . _:b606264094 . _:b606264355 "2"^^ . _:b606264312 . _:b606264354 "2"^^ . _:b51921512 "These niches not only provide sanctuary sites for HSC, but are also co-opted by leukemia cells in hematopoietic malignancies and can support LSC survival 15,>>16<<." . _:b606264315 . _:b606264287 . _:b606264365 "2"^^ . . _:b606264314 . . _:b606264364 "2"^^ . _:b606264157 . _:b606264367 "2"^^ . _:b606264441 . _:b51921544 "Similar to APL, our humanized ossicle model facilitates robust engraftment of primary human MF cells, which has only been of limited success with peripheral blood derived cells with conventional NSG models.31,>>44<< Unlike studies of AML, where disease-initiating cells arise from progenitors 35, we show here that myelofibrosis-initiating cells reside in the HSC compartment, as transplantation of these cells, but not MPP or CMP, results in engraftment" . _:b606264366 "2"^^ . . . _:b606264361 "2"^^ . _:b51921552 "online methods" . _:b606264088 . . _:b606264373 . _:b606264360 "2"^^ . _:b606264213 . _:b606264363 "2"^^ . . _:b51921530 . _:b606264108 . _:b606264362 "2"^^ . . . . _:b51921504 . _:b606264373 "2"^^ . _:b606264405 . _:b606264372 "2"^^ . _:b606264375 "2"^^ . _:b51921514 . _:b606264374 "2"^^ . . . _:b606264369 "2"^^ . . _:b606264109 . . . . _:b606264368 "2"^^ . . _:b606264046 . _:b606264371 "2"^^ . _:b606264370 "2"^^ . . _:b606264418 . _:b606264358 . _:b606264381 "2"^^ . . _:b606264380 "2"^^ . . _:b51921552 _:b51921556 . _:b51921552 _:b51921557 . _:b51921512 . _:b51921552 _:b51921558 . _:b606264383 "2"^^ . _:b51921552 _:b51921559 . _:b51921552 _:b51921553 . . _:b51921552 _:b51921554 . _:b606264382 "2"^^ . _:b51921552 _:b51921555 . . . _:b606264386 . _:b606264377 "2"^^ . . _:b51921552 _:b51921560 . _:b51921552 _:b51921561 . . _:b51921552 _:b51921562 . _:b606264376 "2"^^ . . _:b606264439 . _:b606264379 "2"^^ . . _:b606264431 . _:b606264378 "2"^^ . _:b51921511 "These niches not only provide sanctuary sites for HSC, but are also co-opted by leukemia cells in hematopoietic malignancies and can support LSC survival >>15<<,16." . _:b606264103 . _:b606264389 "2"^^ . _:b606264335 . _:b606264388 "2"^^ . _:b51921554 . _:b606264391 "2"^^ . _:b606264160 . _:b606264298 . . _:b51921544 . . _:b606264390 "2"^^ . . _:b606264215 . . . _:b606264385 "2"^^ . _:b606264187 . _:b606264384 "2"^^ . . . _:b606264052 . _:b51921534 "More recent studies suggested that LIC/LSC are more frequent and can be found in additional immunophenotypically defined subpopulations 7,>>34<<,35. Here, we provide evidence that with a humanized niche, the frequency of LIC is substantially higher than that determined in the NSG mouse BM with the same sample, suggesting that AML LIC frequency is critically dependent on leukemia" . _:b606264387 "2"^^ . . _:b606264386 "2"^^ . _:b606264024 . _:b606264210 . _:b51921509 . _:b606264397 "2"^^ . . _:b606264396 "2"^^ . _:b606264075 . . . _:b606264013 . _:b606264399 "2"^^ . _:b606264398 "2"^^ . . . _:b606264416 . . _:b606264393 "2"^^ . _:b606264392 "2"^^ . . _:b606264123 . _:b606264395 "2"^^ . . _:b606264033 . _:b606264394 "2"^^ . . _:b606264193 . _:b606264405 "2"^^ . _:b606264078 . _:b606264018 . _:b51921523 "Transplantation of human acute promyelocytic leukemia (APL) into immunocompromised mice has so far been of limited success with low to no detectable engraftment >>9<<,23. Since humanized ossicle niches show superior engraftment of non-APL AML samples, we investigated the engraftment of primary human APL cells by direct intraossicle injection." . _:b606264404 "2"^^ . _:b606264267 . _:b606264407 "2"^^ . _:b606264406 "2"^^ . _:b606264255 . _:b51921552 . _:b606264133 . _:b606264401 "2"^^ . . _:b606264400 "2"^^ . . _:b606264403 "2"^^ . _:b51921508 . _:b606264083 . _:b606264402 "2"^^ . . _:b51921511 . _:b606264413 "2"^^ . _:b51921504 "introduction" . . . . _:b606264412 "2"^^ . . _:b606264407 . _:b606264415 "2"^^ . _:b606264044 . _:b606264186 . _:b606264414 "2"^^ . _:b606264034 . . . _:b606264409 "2"^^ . _:b606264380 . . . _:b51921505 "developed, and particularly with the development of NSG mice bearing a targeted deletion of the IL2-receptor gamma chain on the NOD-SCID background, engraftment of many human solid tumors and hematopoietic malignancies became feasible>>1<<. However, human hematopoietic cell engraftment often remained at low levels leading to the development of further strains with improved xenograft efficiency through overexpression or targeted insertion of human cytokines such as SCF," . _:b606264408 "2"^^ . . . _:b51921508 . _:b606264411 "2"^^ . _:b51921518 . . _:b606264410 "2"^^ . . _:b606264245 . _:b606264339 . . _:b606264421 "2"^^ . . . _:b606264420 "2"^^ . _:b51921557 . _:b606264148 . . _:b606264014 "9"^^ . _:b606264423 "2"^^ . _:b606264261 . . _:b606264015 "9"^^ . _:b606264422 "2"^^ . _:b606264009 "17"^^ . _:b606264010 "14"^^ . _:b606264013 "11"^^ . _:b606264417 "2"^^ . _:b606264012 "11"^^ . _:b606264207 . _:b606264416 "2"^^ . _:b606264143 . _:b606264011 "11"^^ . _:b606264194 . _:b606264419 "2"^^ . _:b606264235 . _:b606264418 "2"^^ . _:b606264429 "2"^^ . _:b51921527 . . _:b606264428 "2"^^ . _:b606264209 . _:b51921558 "For single cell transplantation, Lin-CD34+CD38- cells were sorted into single wells of Terasaki plates (Nunc MiniTrays, Thermo Fisher) prefilled with 10 \u03BCL IMDM 10% FBS as previously described >>52<< using FACS AriaII." . . _:b606264022 "8"^^ . _:b606264431 "2"^^ . _:b606264023 "7"^^ . _:b606264156 . _:b606264430 "2"^^ . . _:b606264020 "8"^^ . . _:b606264425 "2"^^ . . . _:b606264021 "8"^^ . _:b606264424 "2"^^ . _:b51921523 . _:b606264018 "8"^^ . . _:b606264427 "2"^^ . _:b606264019 "8"^^ . _:b606264426 "2"^^ . . _:b606264444 . _:b606264016 "9"^^ . _:b606264437 "2"^^ . _:b606264426 . . _:b606264017 "8"^^ . _:b606264436 "2"^^ . _:b606264030 "6"^^ . _:b606264150 . . _:b606264439 "2"^^ . _:b606264031 "6"^^ . _:b606264135 . _:b606264438 "2"^^ . _:b606264070 . _:b606264028 "7"^^ . _:b606264433 "2"^^ .