Culture Of Hematopoietic Stem Cells Research Articles

In vitro Human Bone Marrow Analog: Clinical Potential

Bone marrow is the primary site of hematopoiesis in adult humans. Bone marrow can be cultured in vitro but few simple culture systems fully support hematopoiesis beyond a few months. Human bone marrow analogs are long-term in vitro cultures of marrow stromal and hematopoietic stem cells that can be used to produce cells and products normally harvested from human donors. Bone marrow analog systems should exhibit confluence of the stromal cell populations, persistence of hematopoietic progenitor cells, presence of active regions of hematopoiesis and capacity to produce mature cell types for extended periods of time. Although we are still years away from realizing clinical application of products formed by artificial bone marrow analogs, the process of transitioning this research tool from bench to bedside should be fairly straightforward. The most obvious application of artificial marrow would be for production of autologous hematopoietic CD34(+) stem cells as a stem cell therapy for individuals experiencing bone marrow failure due to disease or injury. Another logical application is for 'blood farming', a process for large-scale in vitro production of red blood cells, white blood cells or platelets, for transfusion or treatment. Other possibilities include production of nonhematopoietic stem cells such as osteogenic stromal cells, osteoblasts and rare pluripotent stem cells. Bone marrow analogs also have great potential as ex vivo human test systems and could play a critical role in drug discovery, drug development and toxicity testing in the future.

Bone Marrow Microenvironment in Fanconi Anemia: A Prospective Functional Study in a Cohort of Fanconi Anemia Patients

Fanconi anemia (FA) is a rare condition due to the genetic inactivation of the FA/BRCA pathway. During childhood, most FA patients display progressive bone marrow failure (BMF), the mechanism of which has not been clarified to date. We analyzed BM mesenchymal stem cells (MSCs) from a series of 20 FA patients with BMF (patient median age 12.5 years old, range 7-34). Expression of FANCD2 and sensitivity to mitomycin C, differentiation capacities, and hematopoiesis-supporting abilities, as well as proliferation, cell senescence, and telomere length were assessed. FA MSCs demonstrated hypersensitivity to mitomycin C compared to control MSCs, as expected for FA cells. FA MSCs had normal immunophenotype, support long-term culture of hematopoietic stem cells (HSCs), and display normal differentiation capacities. Telomere loss during cell aging was similar for FA and control MSCs. However, FA MSCs showed reduced long-term proliferation ability, higher stem cell factor and interleukin-6 levels, and increased expression of senescent-associated beta-galactosidase compared to normal MSCs, suggesting a potential role of the BM microenvironment in long-term BMF.

Direct ex vivo expansion of hematopoietic stem cells from umbilical cord blood on membranes

We have developed a direct ex vivo hematopoietic stem cell (HSC) expansion method involving filtration of umbilical cord blood (UCB) through polyurethane foaming membranes. Most of the red blood cells and mononuclear cells flow through the membranes, but HSCs remain on the membranes. The use of this method reduces the working time to less than 30 min before culture of HSCs, while conventional purification of HSCs from UCB that have been purified by the Ficoll–Paque procedure, followed by magnetic-activated cell sorting, results in loss of cells (i.e., a yield less than 15% of HSCs), damages HSCs, and takes a long time to perform (e.g., 5–8 h). Following filtration of the UCB and washing of the membranes, the membranes can be placed into culture medium where the HSCs can be expanded in a three-dimensional (3D) environment ex vivo, such as the bone marrow niche. Direct ex vivo expansion of HSCs from UCB resulted in an increased number of cells (i.e., 6.6- to 45.7-fold), and the expanded cell populations showed good hematopoietic ability in colony-forming unit assays.

Degradation of hydroxyapatite in vivo and in vitro requires osteoclastic sodium-bicarbonate co-transporter NBCn1

Dissolution of the inorganic bone matrix releases not only calcium and phosphate ions, but also bicarbonate. Electroneutral sodium-bicarbonate co-transporter (NBCn1) is expressed in inactive osteoclasts, but its physiological role in bone resorption has remained unknown. We show here that NBCn1, encoded by the SLC4A7 gene, is directly involved in bone resorption. NBCn1 protein was specifically found at the bone-facing ruffled border areas, and metabolic acidosis increased NBCn1 expression in rats in vivo. In human hematopoietic stem cell cultures, NBCn1 mRNA expression was observed only after formation of resorbing osteoclasts. To further confirm the critical role of NBCn1 during bone resorption, human hematopoietic stem cells were transduced with SLC4A7 shRNA lentiviral particles. Downregulation of NBCn1 both on mRNA and protein level by lentiviral shRNAs significantly inhibited bone resorption and increased intracellular acidification in osteoclasts. The lentiviral particles did not impair osteoclast survival, or differentiation of the hematopoietic or mesenchymal precursor cells into osteoclasts or osteoblasts in vitro. Inhibition of NBCn1 activity may thus provide a new way to regulate osteoclast activity during pathological bone resorption.

The Earliest Stages of Loss of Stem Cell Self-Renewal in-Vivo Is Linked to Upregulated Biosynthesis of “Quiet” Mitochondria and Is Influenced by CXCR4 Activation and STAT3 Gene Deletion.

Abstract 2546Poster Board II-523One attribute of all stem cells is self-renewal (SR). Progress has been made in identifying genes/proteins involved in stem cell SR mechanisms, especially in embryonic stem cells, but very little is known in adult stem cells such as hematopoietic stem cells (HSCs). Mitochondria (Mt) have recently been considered more than an ATP generator and mediator of apoptosis. They act as a kind of scaffold for integrating numerous signals to and from the nucleus and from the extracellular environment, and signals involved in differentiation and cell cycle. They also stabilize microtubules of the mitotic spindle and suppress rotation of the mitotic division plane, potentially influencing asymmetric/symmetric stem cell divisions. Populations of primitive hematopoietic cells with low Mt activity (i.e. Rho123low) are highly enriched in long-term repopulating (LTR)-HSC. Because Mt can suppress mitotic spindle rotation by stabilizing spindle microtubules, potentially influencing asymmetric divisions, we hypothesized that Mt biogenesis/function could be important for mechanisms of SR loss and early-stage differentiation of mouse HSC. We examined Mt mass and Mt membrane potential in primitive mouse CD34lo/neg LINnegSca-1posc-kitpos (CD34-LSK) cells, CD34+LSK cells, and related populations. We note that as the small CD34-LSK cells begin to express CD34, Mt mass significantly increases (2×), while Mt membrane potential does not change in these CD34+LSK cells. The CD34+LSK cells then increase in size without further increases in Mt mass, but with upregulated Mt membrane potential. Because CD34 expression in LSK cells is associated with loss of LTR-HSC (i.e. self-renewal) and retention of only short-term repopulating ability, we propose this previously uncharacterized population of LSK cells with increased numbers/mass of “quiet” Mt may be an important component during initial stages of loss of SR and asymmetric HSC division. We found that primitive LSK cells from mice constitutively expressing an SDF-1α/CXCL12 transgene (TG mice) have shifted ratios of these various mitochondrially distinct populations that may be related to CXCR4 signaling and its known influence on glycolysis and STAT3 activity, both of which can influence Mt metabolism. LSK cells in TG mouse total bone marrow significantly increased from .039+/_.01% to .085+/_.02% (n=6), but they were predominantly CD34+LSK cells. These CD34+LSK cells were composed of two distinct types of cells; small/high-Mt mass and large/high-Mt mass cells. The small LSK cells have lower Mt membrane potential while the large LSK cells had increased Mt membrane potential. Because CXCR4 can signal through STAT3, and because STAT3 can bind to Mt and influence their metabolic activities (i.e. membrane potential), we hypothesized that mice with a tissue-specific targeted gene deletion of STAT3 in hematopoietic cells might result in LSK cells that are defective in upregulating Mt membrane potential, which could shift the proportions of the three mitochondrially distinct populations of LSK cells toward the smaller, Mt mass-low, Mt membrane potential-low type of LSK cell. Bone marrow from targeted STAT3-deleted had LSK numbers increased 50%, but they were composed almost entirely of small, Mt mass-high, membrane potential-low, CD34+ type of LSK cells. This is consistent with our hypothesis and suggests that Mt membrane potential may be important for latter stages of CD34-LSK cell differentiation to more lineage-committed progenitor cells but may not be required for initial stages of SR loss. We propose a model where Mt biogenesis occurs before/during loss of SR in CD34-LSK cells. These Mt are metabolically quiescent (“quiet”), attach to spindle microtubules of dividing cells, and influence spindle orientation resulting in loss of SR (small, Mt mass high, Mt potential low, CD34+LSK cells). Before further differentiation, these cells upregulate Mt metabolism/membrane potential and increase in size to facilitate further expansion and differentiation (large, Mt mass high, Mt potential high, CD34+LSK cells). This model could help explain the observed phenotypes of SDF-1 α/CXCL12 transgenic mice and hematopoietic cell-targeted STAT3 deleted mice. This information may also be useful in the search for strategies to prevent loss of SR capacity of HSCs in culture and expansion of HSCs in-vitro. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic stem cell origin of adipocytes

It has generally been believed that adipocytes are derived from mesenchymal stem cells via fibroblasts. We recently reported that fibroblasts/myofibroblasts in a number of tissues and organs are derived from hematopoietic stem cells (HSCs). In the present study, we tested the hypothesis that HSCs also give rise to adipocytes. Using transplantation of a single enhanced green fluorescent protein-positive (EGFP(+)) HSC and primary culture, we examined generation of adipocytes from HSCs. Adipose tissues from clonally engrafted mice showed EGFP(+) adipocytes that stained positive for leptin, perilipin, and fatty acid binding protein 4. A diet containing rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, significantly enhanced the number of EGFP(+) adipocytes. When EGFP(+) bone marrow cells from clonally engrafted mice were cultured under adipogenic conditions, all of the cultured cells stained positive with Oil Red O and Sudan Black B and exhibited the presence of abundant mRNA for adipocyte markers. Finally, clonal culture- and sorting-based studies of Mac-1 expression of hematopoietic progenitors suggested that adipocytes are derived from HSCs via progenitors for monocytes/macrophages. Together, these studies clarify the current controversy regarding the ability of HSCs to give rise to adipocytes. Furthermore, our primary culture method that generates adipocytes from uncommitted hematopoietic cells should contribute to the studies of the mechanisms of early adipocytic differentiation and may lead to development of therapeutic solutions for many general obesity issues.

Common features of megakaryocytes and hematopoietic stem cells: What's the connection?

Megakaryocytes (Mks) are rare polyploid bone marrow cells whose function is to produce blood platelets. Since the purification and cloning of the major Mk cytokine, thrombopoietin, in 1994, considerable progress has been made in understanding the biology of Mk development. Remarkably, these advances have revealed a number of key features of Mks that are shared with hematopoietic stem cells (HSCs), such as common surface receptors, lineage-specific transcription factors, and specialized signaling pathways. Why there should be such a close connection between these two cell types remains unclear. In this Prospect article, we summarize the data supporting these shared features and speculate on possible teleological bases. In particular, we focus on common links involving developmental hierarchy, endothelial cells, and bone marrow niche interactions. This discussion highlights new data showing close ontologic relationship between HSCs and specialized "hemogenic" endothelial cells during development, and functional overlap between Mks/platelets and endothelial cells. Overall, these findings may be of relevance in the development of techniques for HSC ex vivo culture and/or possible generation of HSCs via somatic cell reprogramming.

Osteopontin is required for natural killer cell development (138.19)

Abstract Natural killer (NK) cells are bone-marrow-derived lymphocytes, which share a common progenitor with T cells. Although a model of NK development from hematopoietic stem cells (HSC) has previously been proposed, the factors and detailed mechanisms driving the differentiation of NK remain to be clearly elucidated. Here we show that osteopontin(OPN) is an important molecule as microenvironmental factor for NK development. The OPN deficient mice evidenced distinct defects in the NK populations in both BM and spleen. However OPN deficient HSC also effectively induced the development of NK cells as control HSC, while the experiment of coculture system and in vivo transplantation system showed that the development of NK cells significantly was defected when HSC culture with OPN absence stromal cells, and transplanted HSC rarely induced into NK cells in OPN deficient mice. Also, the level of OPN expression is much more in stromal cells than the developing NK cells, confirming that OPN is important factor as microenvironment for NK development. The secretion of OPN was induced stimulation with IL15 in the BM stromal cells. The function of IL15, as major factor in NK development, might occurs through OPN which is one of other ways for NK development. . In addition, OPN-driven NK maturation was reduced in T-bet-deficient HPC, suggesting that T-bet is required for OPN-mediated NK development. Collectively, these results show that paracrine OPN signaling drives NK-lineage commitment, thus ultimately promoting NK cell development.

An automated system for delivery of an unstable transcription factor to hematopoietic stem cell cultures

An automated delivery system for cell culture applications would permit studying more complex culture strategies and simplify measures taken to expose cells to unstable molecules. We are interested in understanding how intracellular TAT-HOXB4 protein concentration affects hematopoietic stem cell (HSC) fate; however, current manual dosing strategies of this unstable protein are labor intensive and produce wide concentration ranges which may not promote optimal growth. In this study we describe a programmable automated delivery system that was designed to integrate into a clinically relevant, single-use, closed-system bioprocess and facilitate transcription factor delivery studies. The development of a reporter cell assay allowed for kinetic studies to determine the intracellular (1.4 +/- 0.2 h) and extracellular (3.7 +/- 1.8 h and 78 +/- 27 h at 37 degrees C and 4 degrees C, respectively) half-lives of TAT-HOXB4 activity. These kinetic parameters were incorporated into a mathematical model, which was used to predict the dynamic intracellular concentration of TAT-HOXB4 and optimize the delivery of the protein. The automated system was validated for primary cell culture using human peripheral blood patient samples. Significant expansion of human primitive progenitor cells was obtained upon addition of TAT-HOXB4 without user intervention. The delivery system is thus capable of being used as a clinically relevant tool for the exploration and optimization of temporally sensitive stem cell culture systems.

Numerical Fluid-Dynamic Optimization of Microchannel-Provided Porous Scaffolds for the Co-Culture of Adherent and Non-Adherent Cells

Computational fluid dynamic (CFD) techniques were used to optimize the microenvironment inside scaffolds for hematopoietic stem cell (HSC) culture in a perfusion bioreactor. These matrices are meant to be seeded with adherent bone marrow stromal cells and then co-cultivated with HSCs; the scaffold micro-architecture and the fluid-dynamic conditions have to be optimized to avoid non-adherent stem cells being dragged away while ensuring adequate nutrient supply. The insertion of longitudinal microchannels was tested as a tool to improve perfusion in a homogeneous porous scaffold. Models of microchannel-provided scaffolds, characterized by different values of geometric parameters concerning pores and channels, were built, and numerical fluid-dynamic and oxygen-transfer analyses were carried out. The results of the computations indicated that the microchannels created preferential paths for culture medium flow, causing low shear stresses and drag forces within the pores; meanwhile, they improved oxygen delivery by forcing its penetration into the scaffold bulk. In particular, an 85% porous, 3-mm-thick scaffold with 175-microm-diameter pores was considered; at a constant average drag force guaranteeing stem cell suspension inside this porous bulk, the addition of approximately 260-microm-diameter, 700-microm-spaced channels resulted in 34% higher oxygen partial pressure at the exit (approximately 135 vs 101 mmHg), maintaining a wall shear stress median value of approximately 0.14 mPa. The present work demonstrates the capacity of microchannel-provided scaffolds to ensure suitable conditions for HSC culture and shows that CFD methods are a valuable tool to retrieve significant clues for the design of the culture environment.

Pharmacological Manipulation of the RAR/RXR Signaling Pathway Maintains the Repopulating Capacity of Hematopoietic Stem Cells in Culture

The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However, RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study, we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506, a selective RXR modulator, inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters, suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly, using combinatorial peptide phage display, we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation, and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture.

Mesenchymal stem cells from the Wharton's jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture.

Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow-derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton's jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. Umbilical cord-derived MSCs (UC-MSCs) were cultured from the Wharton's jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. These data suggest the potential therapeutic application of Wharton's jelly-derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation.

Photodynamic treatment with mono‐phenyl‐tri‐(N‐methyl‐4‐pyridyl)‐porphyrin for pathogen inactivation in cord blood stem cell products

Hematopoietic stem cell transplants and culture of hematopoietic progenitor cells require pathogen-free conditions. The application of a method of pathogen inactivation in red blood cells using photodynamic treatment (PDT) was investigated for the decontamination of cord blood stem cell (CBSC) products. CBSC products, spiked with Gram-positive and Gram-negative bacteria, were treated with PDT using mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light. After PDT, in vitro and in vivo evaluation of the CBSC functions were performed. PDT of CBSC products resulted in the inactivation of the bacteria, with Staphylococcus aureus being the most resistant. Complete decontamination was achieved when CBSC products were contaminated with low titers of bacteria. PDT had no effect on white blood cell viability, the ex vivo expansion potential of the progenitor cells, and their capacity to differentiate to various hematopoietic cell lineages. However, PDT reduced the engraftment of human CBSCs in NOD/SCID mice, particularly affecting the B-cell lineage engraftment. Pathogen inactivation of CBSC with Tri-P(4)-mediated PDT is feasible at contamination level up to 10 to 20 colony-forming units per mL and can be considered when ex vivo expansion culture is anticipated. However, this treatment is not recommended for transplantation purposes at this time. Further investigations may elucidate why engraftment is diminished.

Pleiotrophin Is a Growth Factor for Hematopoietic Stem Cells and Induces Stem Cell Self-Renewal

The ability to undergo self-renewal is a defining feature of hematopoietic stem cells (HSCs) but the extrinsic signals which regulate HSC self-renewal remain unclear. We performed a genome-wide expression analysis on primary human brain ECs (HUBECs, n=10) which support the ex vivo expansion of HSCs in non-contact culture (Blood 100:4433–4439; Blood 105:576–583) and non-brain ECs which do not support HSC expansion (n=8) in order to identify soluble proteins overexpressed by the HSC-supportive HUBECs. We identified pleiotrophin (PTN), an 18 kD heparin binding growth factor, to be 32-fold overexpressed in HUBECs as compared to non-supportive EC lines. PTN has established activity in angiogenesis, embryogenesis, neuronal cell growth and tumorigenesis, but has no known function in hematopoiesis. We first tested whether secreted PTN was responsible for the amplification of HSCs that we have observed in co-cultures of HSCs with HUBECs via “loss of function” studies in which a blocking anti-PTN antibody was added to HUBEC cultures and HSC content was measured. Competitive repopulating unit (CRU) assays were performed in which limiting doses of donor CD45.1+ bone marrow (BM) 34−c-kit+sca-1+lin− (34-KSL) HSCs (10, 30 or 100 cells) or their progeny following 7 day non-contact culture with HUBECs + IgG or HUBECs + a blocking anti-PTN were transplanted into lethally irradiated CD45.2+ C57Bl6 mice. Mice transplanted with the progeny of 34-KSL cells cultured with HUBECs demonstrated 4–6 fold increased levels of donor-derived CD45.1+ multilineage repopulation at 8-, 12- and 24-weeks post-transplantation as compared to mice transplanted with input 34-KSL cells. In contrast, mice transplanted with the progeny of 34-KSL cells following culture with HUBECs + anti-PTN demonstrated significant reduction in donor CD45.1+ cell repopulation compared to mice transplanted with the progeny of HUBEC cultures and no difference in donor CD45.1+ cell engraftment compared to mice transplanted with input 34-KSL cells. CRU frequency within day 0 34-KSL cells was estimated to be 1 in 40 cells (95% Confidence Interval [CI]: 1/22-1/72), whereas the CRU estimate within the progeny of 34-KSL cells following HUBEC culture was 1 in 4 cells (CI: 1/2-1/9). The addition of anti-PTN to the HUBEC co-culture decreased the CRU estimate to 1 in 29 cells (CI: 1/16-1/52), suggesting that PTN signaling was responsible for the expansion of HSCs observed in HUBEC co-cultures. In order to confirm whether PTN is indeed a novel growth and self-renewal factor for HSCs, we next performed “gain of function” studies in which 34-KSL cells were placed in liquid suspension cultures with cytokines (thrombopoietin 50 ng/mL, SCF 120 ng/mL, flt-3 ligand 20 ng/mL) with and without the addition of increasing doses of recombinant murine PTN (10, 50 and 100 ng/mL) and total cell expansion and HSC content were compared. The addition of 100 ng/mL PTN to cytokine cultures caused a 20-fold increase in KSL cell content at day 7 compared to input (P<0.001), whereas a decline in KSL cells was observed with cytokine cultures alone (P<0.001), suggesting that PTN caused an expansion of stem/progenitor cells in vitro. Competitive repopulating assays were performed in which CD45.2+ recipient mice were lethally irradiated and transplanted with limiting doses (10, 30 and 100 cells) of CD45.1+ donor BM 34-KSL cells or their progeny following culture with cytokines alone or cytokines + 100 ng/mL PTN. CRU analysis at 4 weeks post-transplantation revealed that the CRU frequency within input 34-KSL cells was was 1 in 32 cells (CI: 1/18-1/57) and the CRU estimate within the progeny of 34-KSL cells cultured with cytokines alone was 1 in 69 (CI: 1/36-1/130). Conversely, the CRU estimate within the progeny of 34-KSL cells cultured with cytokines + PTN was 1 in 4 cells (CI: 1/2-1/10), indicating a 8-fold increase in short term repopulating cell content in response to PTN treatment. Longer term analysis will be performed in these mice to confirm whether PTN treatment induces the self-renewal and amplification of long-term repopulating HSCs in culture. Taken together, these data demonstrate that secreted PTN is primarily responsible for amplification of HSCs that we have observed in cultures of HSCs with ECs and the addition of PTN alone induces the expansion of phenotypic and functional HSCs in culture. PTN is therefore a novel soluble growth factor for HSCs and appears to play an important role in the extrinsic regulation of HSC self-renewal.

Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint–Apoptosis Coupling

Previously, we reported that the spindle assembly checkpoint (SAC), which is coupled in somatic cells, is uncoupled from apoptosis-initiation in mouse and human embryonic stem cells (ESCs). This condition allows ESCs to tolerate and proliferate as polyploidy/aneuploid cells. Proper function of the SAC is vital to prevent polyploidy/aneuploidy during ex vivo hematopoietic stem cell (HSC) expansion. Here we address, for the first time, whether HSCs are more like ESCs or somatic cells with respect to SAC-apoptosis coupling. Using multiparametric permeablized cell flow-cytometric analysis to identify and analyze the mouse sca 1(+)/c-kit(+)/lin(-) (LSK) population, we found the mitotic spindle checkpoint to be functional in primary murine LSK cells, a population enriched in primitive hematopoietic stem/progenitor cells, after prolonged activation of the SAC by microtubule-depolymerizing agents such as nocodazole. HSCs can efficiently initiate apoptosis after activation of the SAC in LSK cells as indicated by increased hypodiploidy and increased levels of activated caspase 3, suggesting that HSCs behave more like somatic cells instead of ESCs with respect to this important cell cycle checkpoint. We conclude that mouse HSCs are not subject to the same kinds of chromosomal instability as are ESCs, knowledge that might aid in optimizing in vitro culture and expansion of human bone marrow or cord blood HSC for clinical applications.

Cytokines regulating hematopoietic stem cell function

Regulation of the multiple fates of hematopoietic stem cells - including quiescence, self-renewal, differentiation, apoptosis, and mobilization from the niche - requires the cooperative actions of several cytokines and other hormones that bind to receptors on these cells. In this review we discuss recent advances in the identification of novel hematopoietic stem cell supportive cytokines and the mechanisms by which they control hematopoietic stem cell fate decisions. Several extrinsic factors that stimulate ex-vivo expansion of hematopoietic stem cells were recently identified by a number of experimental approaches, including forward genetic screening and transcriptional profiling of supportive stromal cells. Recent experiments in which multiple cytokine signaling pathways are activated or suppressed in hematopoietic stem cells reveal the complexity of signal transduction and cell-fate choice in hematopoietic stem cells in vivo and in vitro. The study of genetically modified mice and improvements in the in-vitro hematopoietic stem cell culture system will be powerful tools to elucidate the functions of cytokines that regulate hematopoietic stem cell function. These will further reveal the complex nature of the mechanisms by which extrinsic factors regulate signal transduction and cell-fate decisions of hematopoietic stem cells.

A Real-Time Multi-Channel Monitoring System for Stem Cell Culture Process

A novel, up to 128 channels, multi-parametric physiological measurement system suitable for monitoring hematopoietic stem cell culture processes and cell cultures in general is presented in this paper. The system aims to measure in real-time the most important physical and chemical culture parameters of hematopoietic stem cells, including physicochemical parameters, nutrients, and metabolites, in a long-term culture process. The overarching scope of this research effort is to control and optimize the whole bioprocess by means of the acquisition of real-time quantitative physiological information from the culture. The system is designed in a modular manner. Each hardware module can operate as an independent gain programmable, level shift adjustable, 16 channel data acquisition system specific to a sensor type. Up to eight such data acquisition modules can be combined and connected to the host PC to realize the whole system hardware. The control of data acquisition and the subsequent management of data is performed by the system's software which is coded in LabVIEW. Preliminary experimental results presented here show that the system not only has the ability to interface to various types of sensors allowing the monitoring of different types of culture parameters. Moreover, it can capture dynamic variations of culture parameters by means of real-time multi-channel measurements thus providing additional information on both temporal and spatial profiles of these parameters within a bioreactor. The system is by no means constrained in the hematopoietic stem cell culture field only. It is suitable for cell growth monitoring applications in general.

Oncostatin M-Mediated Regulation of KIT-Ligand-Induced Extracellular Signal-Regulated Kinase Signaling Maintains Hematopoietic Repopulating Activity of Lin−CD34+CD133+ Cord Blood Cells

We investigated whether KIT signaling was sufficient to maintain human hematopoietic stem cells in culture or whether, as with murine stem cells, signaling through glycoprotein 130 (gp130) is additionally required. Sorted CD34(+)CD133(+)(CD33/CD38/CD71)(-) cells from human umbilical cord blood (UCB) were cultured in the presence of combinations of KIT-ligand (KL) and the gp130 stimulating molecule oncostatin M (OSM). We found that OSM increased KL-induced proliferation, which was accompanied by an expansion in numbers of mature progenitors colony-forming cells (CFC, CAFCw2). More primitive progenitors, CAFCw6 and long-term culture-CFC, were not maintained by KL as a single factor. Although addition of OSM did not improve survival, the KL/OSM combination showed improved maintenance of immature progenitors as well as higher CD34 expression. Similarly, both KL and OSM were required to maintain NOD/SCID-repopulating activity. In experiments to investigate the underlying mechanism, we found that extracellular signal-regulated kinase (ERK) and its downstream target p90 ribosomal S6 kinase were activated by KL and downregulated by the inclusion of OSM during stimulation. The p38 mitogen-activated protein kinase (p38 MAPK) was not modulated by either KL or OSM. Indeed, many of the effects of OSM (increased cell division, maintenance of CFC, and maintenance of high CD34 expression) could be mimicked by using the mitogen-activated protein kinase kinase inhibitor U0126. More importantly, NOD/SCID-repopulating activity was preserved in the KL/U0126-stimulated cells, but not in cells stimulated with a combination of KL and the p38 MAPK inhibitor SB203580. Our results show that the loss of repopulating activity during KL stimulation is counteracted by OSM through the downregulation of ERK pathway signaling. Disclosure of potential conflicts of interest is found at the end of this article.

Identification of Pleiotrophin as a Novel Growth Factor for Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem and progenitor cells have been demonstrated to reside in association with bone marrow sinusoidal endothelial cells in vivo but the mechanisms through which the BM vascular niche regulates hematopoiesis remains incompletely defined. In an attempt to identify candidate soluble growth factors for hematopoietic stem cells (HSCs) which are produced by endothelial cells (Ecs), we have performed comparative genome-wide expression analysis of a primary human brain-derived Ecs which we have previously demonstrated to support a 10-fold expansion of human SCID-repopulating cells (SRCs) in non-contact cultures. This analysis revealed that pleiotrophin (PTN), a heparin binding growth factor, was 32-fold overexpressed in HUBECs that supported human HSC expansion as compared to non-supportive EC lines. Pleiotrophin has anti-apoptotic, mitogenic and transforming activities, suggesting a role in tumorigenesis. No role for pleiotrophin in hematopoiesis has been described. In order to determine if PTN contributes to the demonstrated HSC-supportive activity of HUBECs, the activity of PTN was blocked by adminstration of a neutralizing anti-PTN mAb to the coculture model. Three doses of PTN neutralizing antibody (5, 25, and 50 ug/ml) were added to non-contact cocultures of HUBECs with human cord blood CD34+CD38-lin- HSCs. The anti-PTN treatment caused a decrease in the numbers of phenotypic HSCs in culture in a dose dependent manner. Relative to control HUBEC cultures, the % and expansion of CD34(+) progenitors decreased signficantly at the two higher doses of anti-PTN (p<0.05, respectively). Additionally, CD34(+)CD38(−) lin(−) progenitors were decreased by 33% at the highest dose of anti-PTN compared to control HUBEC cultures (p<0.05). Transplantation studies of anti-PTN treated versus control HUBEC-cultured progeny into NOD/SCID mice are ongoing to confirm the central role of PTN in the expansion of human HSCs in culture. In a correlative study, the addition of recombinant PTN to cultures of human HSCs with thrombopoietin, SCF and flt-3 ligand was shown to significantly increase CD34+ cell content and CD34+CD38- cell content compared to cytokines alone, again in a dose-dependent manner. Taken together these results suggest that PTN, a growth factor with no previously ascribed hematopoietic activity, is a soluble growth factor for human hematopoietic progenitor cells. Since blockade of PTN activity significantly inhibits the soluble activity of HUBECs to support human HSC proliferation and expansion, PTN is also a candidate growth factor for further study in the context of understanding the function of the BM vascular niche.

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

Strategies to expand hematopoietic stem cells (HSCs) ex vivo are of key interest. The objective of this study was to resolve if ability of HOXB4, previously documented to induce a significant expansion of HSCs in culture, may extend to other HOX genes and also to further analyze the HOX sequence requirements to achieve this effect. To investigate the ability of Nucleoporin98-Homeobox fusion genes to stimulate HSC self-renewal, we evaluated their presence in 10- to 20-day cultures of transduced mouse bone marrow cells. Stem cell recovery was measured by limiting-dilution assay for long-term competitive repopulating cells (CRU Assay). These experiments revealed remarkable expansions of Nucleoporin98-Homeobox-transduced HSCs (1000-fold to 10,000-fold over input) in contrast to the expected decline of HSCs in control cultures. Nevertheless, the Nucleoporin98-Homeobox-expanded HSCs displayed no proliferative senescence and retained normal lympho-myeloid differentiation activity and a controlled pool size in vivo. Analysis of proviral integration patterns showed the cells regenerated in vivo were highly polyclonal, indicating they had derived from a large proportion of the initially targeted HSCs. Importantly, these effects were preserved when all HOX sequences flanking the homeodomain were removed, thus defining the homeodomain as a key and independent element in the fusion. These findings create new possibilities for investigating HSCs biochemically and genetically and for achieving clinically significant expansion of human HSCs.