Use Of Hematopoietic Stem Cells Research Articles

ROBO4-Mediated Vascular Integrity Regulates the Directionality of Hematopoietic Stem Cell Trafficking

SummaryDespite the use of hematopoietic stem cells (HSCs) in clinical therapy for over half a century, the mechanisms that regulate HSC trafficking, engraftment, and life-long persistence after transplantation are unclear. Here, we show that the vascular endothelium regulates HSC trafficking into and out of bone marrow (BM) niches. Surprisingly, we found that instead of acting as barriers to cellular entry, vascular endothelial cells, via the guidance molecule ROBO4, actively promote HSC translocation across vessel walls into the BM space. In contrast, we found that the vasculature inhibits the reverse process, as induced vascular permeability led to a rapid increase in HSCs in the blood stream. Thus, the vascular endothelium reinforces HSC localization to BM niches both by promoting HSC extravasation from blood-to-BM and by forming vascular barriers that prevent BM-to-blood escape. Our results uncouple the mechanisms that regulate the directionality of HSC trafficking and show that the vasculature can be targeted to improve hematopoietic transplantation therapies.

Novel role of hematopoietic stem cells in immunologic rejection of malignant gliomas

Adoptive cellular therapy (ACT) after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has been largely limited to melanoma due to a lack of reliable methods for expanding tumor-specific lymphocytes from the majority of other solid cancers. We have employed tumor RNA-pulsed dendritic cells (DCs) to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative ACT in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative (MA) conditioning, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed DC vaccines. Hematopoietic stem cells (HSCs), administered for salvage from MA conditioning, rapidly migrated to areas of intracranial tumor growth and facilitated the recruitment of tumor-specific lymphocytes through HSC-elaborated chemokines and enhanced immunologic rejection of intracranial tumors during ACT. Furthermore, HSC transplant under non-myeloablative (NMA) conditions also enhanced immunologic tumor rejection, indicating a novel role for the use of HSCs in the immunologic treatment of malignant gliomas and possibly other solid tumors.

Stem cells and applications: a survey.

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have ability to proliferate and differentiate in different tissues. Different stem cells (SC) with different potential can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue or MSC of Wharton Jelly, which have limited potential, are of interest for clinical applications in regenerative medicine because they are easily separated and prepared and no ethical problems are involved in their use.During the last 10 years, these multipotent cells have generated considerable interest and in particular have been shown to escape allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone, cartilage, diabetes, ophthalmology, urology, liver, organ's reconstruction…).

Stem Cells and Regenerative Medicine: Myth or Reality of the 21th Century.

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Wharton's Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organ's reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC.

Intravenous injection of a foamy virus vector to correct canine SCID-X1

AbstractCurrent approaches to hematopoietic stem cell (HSC) gene therapy involve the collection and ex vivo manipulation of HSCs, a process associated with loss of stem cell multipotency and engraftment potential. An alternative approach for correcting blood-related diseases is the direct intravenous administration of viral vectors, so-called in vivo gene therapy. In this study, we evaluated the safety and efficacy of in vivo gene therapy using a foamy virus vector for the correction of canine X-linked severe combined immunodeficiency (SCID-X1). In newborn SCID-X1 dogs, injection of a foamy virus vector expressing the human IL2RG gene resulted in an expansion of lymphocytes expressing the common γ chain and the development of CD3+ T lymphocytes. CD3+ cells expressed CD4 and CD8 coreceptors, underwent antigen receptor gene rearrangement, and demonstrated functional maturity in response to T-cell mitogens. Retroviral integration site analysis in 4 animals revealed a polyclonal pattern of integration in all dogs with evidence for dominant clones. These results demonstrate that a foamy virus vector can be administered with therapeutic benefit in the SCID-X1 dog, a clinically relevant preclinical model for in vivo gene therapy.

Bancos de cordón umbilical: autonomía versus justicia social

In this paper I analize the advantages and disadvantages of autologous and allogeneic use of hematopoietic stem cells from cord blood and if it is convenient to store them in public or private banks. The aim is to provide arguments for a debate that reflects fundamental moral issues of health systems, the rights and responsibilities of citizens in liberal democracies, the commitments we have with the generational equity and the property issues of human tissues.

Modalities to Improve Cord Blood Engraftment

Umblical cord blood (UCB) is one of the major sources of Hematopoietic Stem Cell Transplantation (HSCT) with increasing use in clinical practice. UCB can be a life saver for patients who do not have a matched unrelated adult donor or for patients who are in need of an urgent transplantation. Various factors make UCB a significant source of Hematopoietic Stem Cells (HSCs), including ease of procurement and lack of donor attrition, with the ability to process and long term storage of donor cells. Importantly, UCB donations can be used right away without the need for a “perfect” HLA match, thereby increasing donor access to HSCT, particularly for minority and mixed ethnicity patients, for whom a suitably matched related or unrelated donor may be difficult to locate. The major limitation of UCB is the quantity of cells to be infused. Although high proliferative potential allows one log less use of HSCs (HSC<105/kg) compared to bone marrow (BM) or peripheral blood mononuclear cells (PBSC), even this amount cannot be reached in the majority of patients under donor search. When total nucleated cell (TNC) and CD34+ cell doses in UCB grafts are analyzed, a high correlation is observed with the rate of neutrophil and platelet engraftment, the incidence of graft failure and early transplant-related complications. It has been shown that UCB grafts with more than 3/6 HLA mismatches and with cell doses under the defined minimum threshold lead to higher transplantation related mortality (TRM). Especially when adult cord blood transplantation (UCBT) is the subject, providing units with enough cells remains the major drawback. Despite certain efforts, there is still an unmet need for increasing HSC cell dose and/or stimulating engraftment without loss of their long term repopulating (LTR) potential and reducing graft versus host disease (GVHD). Intrinsic and extrinsic cellular factors have been proven to act roles in HSC expansion thus justifying their role in in vitro and ex vivo culture conditions. Attempts to regulate these factors through ex vivo expansion methods aim to overcome insufficient cell numbers while methods promoting HSC homing is in favor of the latter. Combination of both appear to work synergistically. Induction or adoptive transfer of UCB derived immune cells particularly Natural Killer (NK) cells and regulatory T cells (T reg) with or without cytokines are also effective approaches for gaining better engraftment levels after UCBT. All of these approaches have been denoted as “successful” in pre-clinical in vitro and animal studies. Many of them have also been tested in early/later phase clinical trials resulting in encouraging results. We aim to review the current knowledge on UCB expansion and engraftment enhancer methods, a very rapidly improving field particularly in the last decade.

Manipulation of Hematopoietic Stem Cells for Regenerative Medicine

Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and to differentiate into all blood cell lineages while retaining robust capacity to regenerate hematopoiesis. Based on these characteristics, they are widely used for transplantation and gene therapy. However, the dose of HSCs available for use in treatments is limited. Therefore, extensive work has been undertaken to expand HSCs in culture and to produce HSCs from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in order to improve the efficiency and outcome of HSC-based therapies. Various surface markers have been characterized to improve the purification of HSCs and a huge number of cytokines and small-molecule compounds have been screened for use in the expansion of HSCs. In addition, attempts to generate not only HSCs but also mature blood cells from ESCs and iPSCs are currently ongoing. This review covers recent approaches for the purification, expansion or production of human HSCs and provides insight into problems that need to be resolved.

Culture Of Mobilized Rhesus Macaque CD34+ Cells In Hypoxic Conditions Does Not Improve Lentiviral Transduction Efficiency In Long-Term Repopulating Hematopoietic Stem Cells

Low oxygen levels have been proposed to play a fundamental role in the maintenance of normal hematopoietic stem cells (HSC) function. We investigated whether culture of rhesus macaque mobilized CD34+ cells under low oxygen conditions (1-5% O2) could improve lentiviral transduction efficiency in HSCs compared to culture under atmospheric O2 conditions (21%). Mobilized CD34+ cells from 4 animals were prestimulated for 12 hours in the presence of cytokines and subsequently divided in two equal fractions for transduction with SIN-lentiviral vectors (MOI=100) expressing GFP or TdTomato fluorescent proteins for 48 hours. In 3 animals, cells were transduced with GFP vectors in hypoxia and TdTomato vectors in normoxia. These conditions were reversed in one animal. A portion of the transduced cells was used for phenotypic, cell cycle, clonogenic and apoptosis assays. The remaining cells from both fractions were combined and injected intravenously in lethally irradiated rhesus macaques and in vivo gene marking was measured in peripheral blood granulocytes for up to 21 months after transplantation. The numbers of total live cells and CD34+ cells after 2.5 days in culture were not significantly different compared to baseline for both hypoxic and normoxic conditions (p=0.85 and p=0.84, respectively). Similarly, numbers of CD34+CD38- cells were stable compared to baseline in hypoxia and normoxia (p=0.94). After 2.5 days of culture, the total number of colony-forming cells increased 1.4-fold under both hypoxic and normoxic conditions (p=0.69) compared to freshly isolated CD34+ cells. At baseline, the majority (50.6 + 4.4%) of the CD34+ cells were in the G0 phase of the cell cycle. After 2.5 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 19.9 + 8.8% and 18.6 + 5.9%, respectively (p=0.82). The differences in percentages of cells in the G1 and S/G2/M phases of the cell cycle were also not statistically significant (p=0.175 and p=0.732, respectively). The pO2 had no impact on cell death (18.2 + 5.3% in hypoxia and 16.8 + 4.8% in normoxia, p=0.69) or on the percentages of apoptotic cells (11.1 + 6.7% in hypoxia and 8.4 + 2.3% in normoxia, p=0.47). When considering the bulk of CD34+ cells after transduction with lentiviral vectors, the percentages of GFP-marked cells were consistently higher (range 1.8 to 2.2-fold, mean 2.1-fold) compared to TdTomato-marked cells independently of the transduction conditions used, consistent with a 2.1-fold intrinsic superiority of GFP-based lentiviral vectors compared to TdTomato vectors at equivalent MOI. Taking into account the inherent vector differences, transduction efficiencies were similar in normoxia (70.7 + 11.7%) and hypoxia (68.0 + 16.3%) (p=0.80). When adjusted for the intrinsic superiority of GFP-based vectors, the contribution to long-term in vivo gene marking measured by flow cytometry and quantitative PCR was comparable between cells transduced in 21% pO2 (9.0 + 4.5%) and 5% pO2 conditions (8.4 + 4.9%) (p=0.87). Consistent with these data, GFP and TdTomato-labeled hematopoietic cells were equally visualized, using a confocal/2-photon hybrid microscopy approach, in BM biopsy specimens collected at various time points after transplantation. Given recent evidence suggesting that lower oxygen concentrations (<1.5%) may be required to stabilize HIF-1a, a key sensor of hypoxic conditions, transduction was performed under extreme hypoxic conditions (1% O2) in one animal. While the overall viability of the bulk CD34+ cells after 2.5 days of transduction under extreme hypoxia was comparable to cells cultured in normoxia, their contribution to long-term in vivo marking was negligible (0.3%) compared to cells transduced under normoxic conditions (2.4%), suggesting toxicity of very low oxygen levels on HSCs. Overall, when current methodologies used for the genetic manipulation of HSCs for gene therapy applications were performed under hypoxic conditions, susceptibility to lentiviral transduction of CD34+ cells was not ameliorated compared to cells cultured in 21% O2 conditions in the rhesus macaque transplantation model. The short culture times (2-3 days) used in recent lentivirus-based gene therapy clinical trials and replicated in this study may not be sufficient to impact the phenotype of long-term repopulating HSCs and do not warrant incorporation of hypoxia in current gene therapy protocols. Disclosures:No relevant conflicts of interest to declare.

Developmental changes in hematopoietic stem cell properties

Hematopoietic stem cells (HSCs) comprise a rare population of cells that can regenerate and maintain lifelong blood cell production. This functionality is achieved through their ability to undergo many divisions without activating a poised, but latent, capacity for differentiation into multiple blood cell types. Throughout life, HSCs undergo sequential changes in several key properties. These affect mechanisms that regulate the self-renewal, turnover and differentiation of HSCs as well as the properties of the committed progenitors and terminally differentiated cells derived from them. Recent findings point to the Lin28b-let-7 pathway as a master regulator of many of these changes with important implications for the clinical use of HSCs for marrow rescue and gene therapy, as well as furthering our understanding of the different pathogenesis of childhood and adult-onset leukemia.

MiR-15a inhibits cell proliferation of pancreatic ductal adenocarcinoma (PDAC) by downregulating BMI-1 expression

The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSC) from loss-of-function analysis. Here, we demonstrate that increased expression of Bmi-1 promotes HSC self-renewal. Forced expression of Bmi-1 enhanced symmetrical cell division of HSCs and mediated a higher probability of inheritance of stemness through cell division. Correspondingly, forced expression of Bmi-1, but not the other PcG genes, led to a striking ex vivo expansion of multipotential progenitors and marked augmentation of HSC repopulating capacity in vivo. Loss-of-function analyses revealed that among PcG genes, absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal. Our findings define Bmi-1 as a central player in HSC self-renewal and demonstrate that Bmi-1 is a target for therapeutic manipulation of HSCs.

LOW Tranplant Related Mortality and LOW GRAFT Rejection After Related Haploidentical STEM CELL Transplantation (HAPLO-SCT) Prepared with NON Myeloablative CONDITIONING REGIMEN (NMA) and Posttransplant Cyclophosphamide for Advanced Lymphoid Malignancies.

AbstractAbstract 3031 Introduction:The use of hematopoietic stem cells from partially matched family donors has become an alternative transplant option for patients with no HLA identical donor available. It might be even a first choice, especially in advanced hematological maligancies, because of the presumed stronger graft versus tumor effect relied to the partial HLA identity. However, haplo-SCT is associated with high risk of graft rejection (GR) and severe graft versus host disease (GvHD), especially after non NMA. High dose posttransplant Cyclophosphamide (Cy) has been shown to prevent GvHD and GR. Aim:This retrospective analysis report the outcome of 15 patients (pt) using posttransplant Cy after a NMA in our center. Results:Fifteen adult pt (Male=9) with high risk lymphoid malignancies for whom a HLA matched related or unrelated donor were not available, underwent haplo-SCT from February 2011 to June 2012. The median pt age was 41years (y) (range, 20–60). Diagnosis were Hodgkin Lymphoma in 5 pt, Non Hodgkin Lymphoma in 4 pt, Chronic Lymphatic Leukemia in 2 pt, Multiple Myeloma in 2 pt and Acute Lymphoblastic Leukemia in 3 pt. All pt were heavily pretreated with a median of 3 or more chemotherapy regimens including failure of previous autologous (12pt) or allogeneic transplantation (3pt). At time of transplant 9pt (60%) were in complete remission (CR1=6, CR2=3). The NMA consisted in Fludarabine 30mg/m2 iv on days -6 to -2, Cyclophospamide 14.5mg/kg iv from days -6 and -5 and Total Body Irradiation 200cGy on day -1. For Graft versus Host Disease prophylaxis all pt received postransplant Cy 50mg/kg iv on days + 3 and +4 with Mesna. One day later, all pt started with Cyclosporine (CSA) 3mg/kg/day and Mycophenolatemofetil (MMF) 15mg/kg/day and Filgrastim 5ug/kg/day until neutrophil recovery. MMF was stopped at day+35 and CSA was given until day +180 in the absence of GvHD. Ten pt (67%) received bone marrow (BM) and 5 pt (33%) G-CSF mobilized peripheral blood stem cells (PBSC) as graft source. All donors were HLA haploidentical first degree family donors (Male=8) including brothers (7), sisters (2), mothers (4) and son (1). Median donor age was 49 y (range, 23–66). Pt had HLA typing at the allele or antigen level (A, B, Cw, DRB1, DQ) whereas 8 pt were mismatched at 5 loci, 1 pt at 4 loci, 4 pt at 3 and 2 pt at 2 loci. BM grafts contained a medium of 2×106/kg CD34+ cells and 22×106/kg CD3+ cells. PBSC grafts contained a medium of 6×106/kg CD34+ cells and 298×106/kg CD3+ cells. The engraftment rate was 93%, with a median time to neutrophil recovery (>500 G/l) of 20.5 days (range, 14–26) and to platelet recovery (>20 000 G/l) of 28.5 days (range, 14–41). Compared to BM recipients, pt receiving PBSC experienced faster neutrophil [(17 days (range, 14–23) vs 21 days (range, 18–26), p=0.04] and platelet recovery [(23 days (range, 14–30) vs 30 days (range, 23–41), p=0.04)]. One pt with ALL did not engraft and died at day +40 for infectious complication. One pt died of treatment related mortality with a cumulative incidence of 7% [96%CI (0–20)]. Two pt developped acute GvHD grade 2. No chronic GvHD was observed in pt surviving beyond day 100. Four pt exerienced relapse or progression of the underlying disease. After a median follow up of 197 days (range, 40–518) the actuarial overall survival and event-free survival at 2 years were 87% [95% CI (70–100)] and 51 % [95% CI (16–85)], respectively. Conclusion:NMA haplo-SCT with postransplant Cy in pt with advanced lymphoid malignancies is associated with low NRM, low acute GvHD and low graft rejection. However, better disease control in selected pt is needed to overcome early relapse or progression. Even if follow-up is short, PBSC seems to be an promising option to bone marrow as stem cell source. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Intermittent in Vivo Parathyroid Hormone (PTH) Treatment Results in Jagged 1 (Jag 1) Dependent and Independent Effects On Hematopoietic Stem Cells (HSCs)

Abstract 642HSCs are rare immature cells capable of reconstituting all blood cell lineages throughout the life of an individual. We have previously shown that intermittent treatment with PTH is sufficient to increase the number of HSCs in the marrow of mice. This PTH effect is blocked in vitro with inhibition of gamma-secretase, the mediator of a required step in Notch signaling. Osteoblastic cells are a critical component of the HSC niche and are likely mediators of the PTH-induced increase in HSCs. Specifically the Notch ligand Jag 1 is expressed on osteoblasts and is therefore implicated as a mechanism through which PTH acts on HSCs. Therefore we investigated in vivo the role of osteoblastic Jag 1 in the PTH-dependent increase in HSCs. We utilized the 2.3kb collagen 1 promoter driven cre recombinase to specifically excise Jag 1 from osteoblastic cells in mice (OBJag1 mice). As we previously reported treatment of wild type (WT) controls with PTH 3 times daily for 10 days resulted in a significant increase in phenotypic HSC populations including Lin-Sca1+cKit+CD48-CD150- short-term HSCs (ST-HSCs) (VEH/PTH 0.0405±0.001 vs 0.0650±0.0038, p≤0.0001) and Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (LT-HSCs) (VEH/PTH 0.0077±0.0008 vs 0.0125±0.00096, p≤0.01) as determined by flow cytometric analysis. In contrast treatment of OBJag1 mice did not result in a phenotypic increase in these populations. Despite the lack of a phenotypic increase in HSCs in OBJag1 mice, when HSC function was assessed by competitive repopulation assay, OBJag1 marrow cells demonstrated the same increased repopulating ability as WT mice (WT: VEH/PTH 12.16±2.7 vs 22.32±2.4, p≤0.01, OBJag1: VEH/PTH 13.6±1.8 vs 31.6±5.9, p≤0.01). Upon secondary transplantation however, HSCs from OBJag1 donors treated with PTH resulted in a lower engraftment rate than VEH treated controls (VEH/PTH 14.61±3.8 vs 4.38±0.9, p≤0.05). This result suggests that osteoblastic Jag 1 is necessary for the increase in phenotypic HSCs resulting from PTH treatment and is required to maintain LT-HSC self-renewal. However these data also suggest an osteoblastic Jag 1 independent mechanism that mediates a transient increase in repopulating ability. Decreased apoptosis is a potential mechanism by which PTH may functionally increase HSCs in the absence of increased self-renewal. To determine if PTH treatment decreases the apoptosis rate of HSCs, WT mice were treated intermittently with PTH once a day for 7 days. Despite a lack of increased HSCs by phenotypic analysis at 7 days, marrow from PTH treated mice displayed an increase in LT-HSC function as measured by competitive transplantation. We determined to measure the effect of PTH on apoptotic rates of HSCs using Annexin V membrane expression. By the 7th day of PTH treatment, LT-HSC apoptotic rates were decreased in the PTH treated group (VEH/PTH 10.482±2.25 vs. 6.27±1.93, p≤0.01) suggesting that changes in apoptotic rate of LT-HSCs precedes the HSC increase. These results were confirmed by flow cytometric measurement of activated caspase 3. PTH treatment decreased the percentage of LT-HSCs that were positive for activated caspase 3 (VEH/PTH 4.3±0.5 vs. 2.4±0.3, p≤0.01). PTH induced micro-architectural changes in trabecular bone at day 7 of treatment suggesting bone involvement despite the lack of an increase in bone volume. These results suggest for the first time that PTH may exert its beneficial effect on bone marrow reconstitution through both Jag 1 dependent and independent effects. Additionally, HSCs demonstrate decreased apoptotic rates and increased reconstitution ability prior to a demonstrable phenotypic increase, mimicking the effect seen in the absence of osteoblastic Jag 1. Together these results suggest that the decreased apoptotic rate may be mediated by an osteoblastic Jag 1 independent mechanism. Whether osteoblasts are required for the observed osteoblastic Jag 1 independent effects remains to be seen as these effects could be mediated by a Jag 1 independent osteoblastic mechanism or by an altogether different cellular component of the HSC niche. Further, since stressful manipulation of HSCs ex vivo is essential for their use in transplantation, defining factors regulating and decreasing their apoptosis may improve their engraftment efficiency, expanding their clinical use when their numbers are limited. Disclosures:No relevant conflicts of interest to declare.

MicroRNAs as Markers for Neurally Committed CD133+/CD34+ Stem Cells Derived from Human Umbilical Cord Blood

Neural differentiation of the CD133+/CD34+subpopulation of human umbilical cord blood stem cells was investigated, and neuro-miR (mir-9 and mir-124) expression was examined. An efficient induction protocol for neural differentiation of hematopoietic stem cells together with the exclusion of retinoic acid in this process was also studied. Transcription of some neural markers such as microtubule-associated protein-2, beta-tubulin III, and neuron-specific enolase was evaluated by real-time PCR, immunocytochemistry, and western blotting. Increased expression of neural indicators in the treated cells confirmed the appropriate neural differentiation, which supported the high efficiency of our defined neuronal induction protocol. Verified high expression of neuro-miRNAs along with neuronal specific proteins not only strengthens the regulatory role of miRNAs in determining stem cell fate but also introduces these miRNAs as novel indicators of neural differentiation. These data highlight the prominent therapeutic potential of hematopoietic stem cells for use in cell therapy of neurodegenerative diseases.

Ex vivo erythrocyte generation and blood doping.

Ex vivo erythrocyte generation and blood doping.

Concise Review: Hematopoietic Stem Cell Aging, Life Span, and Transplantation

Self-renewal and multilineage differentiation of stem cells are keys to the lifelong homeostatic maintenance of tissues and organs. Hematopoietic aging, characterized by immunosenescence, proinflammation, and anemia, is attributed to age-associated changes in the number and function of hematopoietic stem cells (HSCs) and their microenvironmental niche. Genetic variants and factors regulating stem cell aging are correlatively or causatively associated with overall organismal aging and longevity. Translational use of HSCs for transplantation and gene therapy demands effective methods for stem cell expansion. Targeting the molecular pathways involved in HSC self-renewal, proliferation, and homing has led to enhanced expansion and engraftment of stem cells upon transplantation. HSC transplantation is less effective in elderly people, even though this is the demographic with the greatest need for this form of treatment. Thus, understanding the biological changes in the aging of stem cells as well as local and systematic environments will improve the efficacy of aged stem cells for regenerative medicine and ultimately facilitate improved health and life spans.

Bone, microenvironment and hematopoiesis

Hematopoietic stem cells (HSCs) mature to form all blood cells and the study into how HSC fate decisions are made has exploded in recent years. In an effort to fully understand the function and organization of HSCs and hematopoietic progenitor cells (HPCs), many groups have identified the microenvironment in which they reside as playing a key role. This review highlights the findings within the last 18 months on the cells and molecules shown to be important within the bone marrow HSC niche for HSC regulation. Previous research has heavily concentrated on the role of osteoblasts, mesenchymal stem cells (MSCs), reticular stromal cells, endothelial cells and nerve cells. More recently, research has not only expanded on the role of these cells, but has also shown that mature hematopoietic cells such as macrophages and megakaryocytes are also important in the maintenance of hematopoiesis within the HSC niche. Identifying and understanding the roles of all cells comprising the HSC niche coupled with the development of better 3D imaging and 3D in-vitro mimicking of the HSC niche will increase our understanding of where HSCs reside and how they are regulated. Research will lead to better manipulation of HSCs for mobilization, homing and hematopoietic reconstitution following injury or disease.

The secrets of the bone marrow niche: Enigmatic niche brings challenge for HSC expansion

The bone marrow niche keeps puzzling scientists in cancer and regenerative medicine. What elements constitute the niche and how it affects neighbor cells in different disease contexts remain to be a matter of debate and extensive investigation. The translational use of hematopoietic stem cells (HSCs) in transplantation biology poses a challenge, given the propensity of these cells to remain quiescent. Although the niche is a good candidate to exploit for reprogramming HSCs and controlling their expansion, new studies have added to its complexity. In 'Bench to Bedside', Paul S. Frenette and Yuya Kunisaki examine these studies to discuss how new players and their signals are involved in HSC maintenance and what the implications are for the development of HSC-based therapies. Among the alterations occurring in leukemias, metabolic events seem to foster cancer progression but may also be involved in cancer predisposition. Rushdia Z. Yusuf, Ying-Hua Wang and David T. Scadden peruse recent clinical and experimental studies that look at myelodysplastic syndromes and secondary leukemias and argue how metabolic changes in these cancers may not only be cell autonomous but also can emanate from the bone marrow stroma. Targeting this niche may open new avenues to reduce the risk for secondary leukemias in cancer survivors.

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.