Hematopoietic Inductive Microenvironment Research Articles

Reconstruction of Hematopoietic Inductive Microenvironment after Transplantation of VCAM-1-Modified Human Umbilical Cord Blood Stromal Cells

The hematopoietic inductive microenvironment (HIM) is where hematopoietic stem/progenitor cells grow and develop. Hematopoietic stromal cells were the key components of the HIM. In our previous study, we had successfully cultured and isolated human cord blood–derived stromal cells (HUCBSCs) and demonstrated that they could secret hemopoietic growth factors such as GM-CSF, TPO, and SCF. However, it is still controversial whether HUCBSCs can be used for reconstruction of HIM. In this study, we first established a co-culture system of HUCBSCs and cord blood CD34+ cells and then determined that using HUCBSCs as the adherent layer had significantly more newly formed colonies of each hematopoietic lineage than the control group, indicating that HUCBSCs had the ability to promote the proliferation of hematopoietic stem cells/progenitor cells. Furthermore, the number of colonies was significantly higher in vascular cell adhesion molecule-1 (VCAM-1)-modified HUCBSCs, suggesting that the ability of HUCBSCs in promoting the proliferation of hematopoietic stem cells/progenitor cells was further enhanced after having been modified with VCAM-1. Next, HUCBSCs were infused into a radiation-damaged animal model, in which the recovery of hematopoiesis was observed. The results demonstrate that the transplanted HUCBSCs were “homed in” to bone marrow and played roles in promoting the recovery of irradiation-induced hematopoietic damage and repairing HIM. Compared with the control group, the HUCBSC group had significantly superior effectiveness in terms of the recovery time for hemogram and myelogram, CFU-F, CFU-GM, BFU-E, and CFU-Meg. Such differences were even more significant in VCAM-1-modified HUCBSCs group. We suggest that HUCBSCs are able to restore the functions of HIM and promote the recovery of radiation-induced hematopoietic damage. VCAM-1 plays an important role in supporting the repair of HIM damage.

Effects of Bone Marrow Stromal Cells and Umbilical Cord Blood-Derived Stromal Cells on Daunorubicin-Resistant Residual Jurkat Cells

Objective To observe the effects of the hematopoietic inductive microenvironment (HIM) simulated by stromal cells of different origins on daunorubicin-resistant residual Jurkat cells (Jurkat/DNR cells). Methods Jurkat/DNR cells were cultured and identified. Human umbilical cord blood-derived stromal cells (UCBDSCs) and normal human bone marrow stromal cells (BMSCs) were isolated and cocultured with Jurkat/DNR cells. Jurkat/DNR cells were collected after 14 days of coculture and analyzed with regard to cell proliferation and differentiation abilities, apoptosis, drug sensitivity, and MRD1 multidrug resistance gene mRNA expression. Results UCBDSC-simulated HIM suppressed proliferation and promoted apoptosis, differentiation, and drug sensitivity of Jurkat/DNR cells more significantly than BMSC-simulated HIM. Conclusions Both BMSCs and UCBDSCs reconstruct the leukemic HIM and reverse drug resistance in Jurkat/DNR cells. UCBDSCs reconstruct the leukemic HIM and reverse drug resistance more significantly than BMSCs.

Bone Marrow Mesenchymal Stem Cells: Historical Overview and Concepts

This review describes the historical emergence of the concept of bone marrow mesenchymal stem cells (MSCs), summarizing data on Wolf and Trentin's hematopoietic inductive microenvironment; Dexter's hematopoiesis-supportive stromal cells; Friedenstein's osteogenic cells; and Pittenger's trilineal osteoblastic, chondrocytic, and adipocytic precursors; to finally introduce the specific bone marrow mesenchymal stem cells with differentiation potential to four lineages (mesenchymal and vascular smooth muscle lineages), and stromal and immunomodulatory capacities. Two points are the object of detailed discussion. The first point envisions the stem cell attributes (multipotentiality, self-renewal, tissue regeneration, population heterogeneity, plasticity, and lineage priming) compared with that of the paradigmatic hematopoietic stem cell. In the second point, we discuss the possible existence of bone marrow cells with greater differentiation potential, eventually pluripotential cells. The latter point raises the issues of cell fusion, reprogramming, or selection under nonstandardized conditions of rare populations of neuroectodermal origin, or of cells that had undergone mesenchymal-to-epithelial transition. In the last section, we review data on MSC senescence and possible malignant transformation secondary to extensive culture, gene transfer of telomerase, or mutations such as leading to Ewing's sarcoma. The set of data leads to the conclusion that bone marrow MSCs constitute a specific adult tissue stem cell population. The multiple characteristics of this stem cell type account for the versatility of the mechanisms of injured tissue repair. Although MSC administration may be extremely useful in a number of clinical applications, their transplantation is not without risks that must not be overlooked when developing cell therapy protocols.

Long-Term in Vitro Cytokine-Free and Serum-Free Culture of Human Cord Blood Mononuclear Cells in a Three-Dimensional Scaffold.

Abstract 503The ability to expand cord blood (CB) cells ex vivo overcomes an important limitation to its wider clinical application in cellular therapies. The current practice of hematopoietic cell culture is based on two-dimensional (2D) tissue culture flasks or well plates which require either co-culture with allogeneic or xenogeneic stromal cells and the exogenous provision of several cytokines. This 2D culture environment is artificial and lacks the 3D cellular niches that characterise the in vivo hematopoietic inductive microenvironment. Specifically, the cultured cells are exposed to abnormally high cytokine concentrations, which may result in differentiation and loss of pluripotency.We have previously developed a 3D bone marrow biomimicry through the use of synthetic scaffolds made of poly (D,L-lactide-co-glycolide) (PLGA) and polyurethane (PU) coated with collagen type I. Our previous work has shown that these scaffolds, which were seeded with cord blood (CB) mononuclear cells (MNCs) at a cell density of 3-6×106cells per scaffold (5×5×5mm3), could successfully support long-term culture in the absence of exogenous growth factors for over 4 weeks. Specifically, the 3D biomimicry facilitated a 53-fold total MNC expansion, with an increase in the BFU-E and CFU-GM progenitor cell population. However, these cultures, although cytokine-free, contained 20-30% (v/v) fetal calf serum which can have both conducive and inhibitory effects on hematopoietic cell cultures due to the unknown composition and concentration of humoral factors contained within. Inclusion of serum in expansion-type cultures can limit the clinical application of the derived product.The serum-free and cytokine-free culture and expansion of hematopoietic cells has not been achieved until now. Herein, we report that for at least 4 weeks the polyurethane (PU) scaffolds coated with collagen type I were able to maintain and expand human CB MNCs. Furthermore the progenitor population, as determined by the colony forming unit assay, was also maintained and preferentially directed towards the granulocytic lineage, even though the CFU-GEMMs declined. Immunophenotypic analysis of the extracted cells confirmed the presence of erythroid precursors (CD71+CD45-) as well as early maturing myeloid cells. In contrast, the 2D cytokine- and serum-free cultures collapsed within 3-4 days. We hypothesized that the 3D biomimicry was able to facilitate serum- and cytokine-free conditions because it can recapitulate the three-dimensional architecture of the human bone marrow. This hypothesis was supported by scanning electron microscopy of the central sections of the scaffolds that showed the migration of cells within the pores and establishment of “niche-like” structures. In conclusion, this novel 3D culture system is capable of long-term, cytokine- and serum-free expansion of haematopoietic cells from cord blood, enabling the study of haematopoiesis as well as facilitating the expansion of cells for future clinical applications. Disclosures:No relevant conflicts of interest to declare.

Expansion of Hematopoietic Stem Cells (HSC) from Cord-Blood (CB) Derived Mononuclear Cells (MNC) in Cytokine-Free Environment Using Mesenchymal Cells Spatial Co-Culture System.

In situ, HSCs are intimately associated with discrete spatially organized niches within the bone marrow, which are part of the “hematopoietic inductive microenvironment” (HIM). The HIM provides a range of molecular signals that collectively control HSC differentiation and self-renewal. This process is mediated via cell-cell and cell-ECM molecular contacts or through specific factors synthesized and secreted by mesenchymal stromal cells (MSC).Current strategies aimed at ex vivo expansion of transplantable HSC have so far been met with limited success. Most attempts to expand HSC ex-vivo are based on using monolayers of MSC as a supportive tier or growth media supplemented with blend of cytokines. failure to support long-term maintenance and expansion of human HSC on MSC monolayers could be associated with inadequate physical architecture of the culture systems which does not reflect the natural 3-D growth conditions present within the BM-HIM. The other method based on supporting the Hematopoietic cells proliferation by a blend of cytokines, is proven routine but post transplantation marrow replenishment is currently unsatisfactory and chromosomal epigenetic modifications may be introduced into progeny cells.Using the PluriX™ bioreactor, we had previously demonstrated that spatial co-cultures of MSC and HSCs provide an efficient system for the expansion of HSCs from CB CD34+ selected cells in cytokine-free environment.Over the past years, attempts to expand HSCs mostly employed CD34+ selected cells. However, these cells may not represent the earliest HSCs and the immunomagnetic selection protocols are costly, time consuming and associated with great loss of source cells. A theoretical approach to overcome both hurdles is by using non-selected MNC as the founding population of HSC. Following our ability to expand HSCs from CD34+ selected cells, we now demonstrate the capacity of the 3-D HSC MSCs co-culture system within the PluriX™ bioreactor system to expand HSCs using MNC as HSCs source.Primary Human marrow-derived MSCs were grown on 3-D carriers within the PluriX™bioreactor system. When the MSCs cultures reached high density (3*106–8*106 cells/ml) CD34+ selected cells or MNC were plated onto them. Within 7–14 days, the population of CD34+ and CD34+CD38− cell were expanded irrespective of the HSCs founding source. However, during this period of time the absolute expansion magnitude was greater when MNC rather than CD34+ selected cells were used to drive the process. The enrichment rate of CD34+cell from MNC and CD34+ selected cells was 20–100 vs. 5–20 fold, respectively. Under same conditions, the enrichment of the earlier CD34+CD38− cells was 40–50 vs. up to 10 fold for the same cell populations. In-conclusion, the spatial co-cultures of MSC and HSC within the PluriX™ bioreactor have been shown to form a potent HSCs expansion system in non-supplemented cytokine environment. The capacity of this practice is improved when MNCs, instead of CD34+ selected cells, serve as the originating population for HSCs. It is conceivable that the efficiency of this system is based upon selective adherence between HSCs and the hematopoietic niches within the MSCs 3-D cultures. As such, upscale of the current system could become attractive method for HSCs expansion.

Pathology and treatment of anemia in the elderly

An important background characteristic of anemia in the elderly is decrease in hematopoiesis due to aging. Factors influencing hematopoiesis in the elderly include changes in the distribution of hematopoietic tissue, changes in hematopoietic stem cell density and changes in the hematopoietic inductive microenvironment. In the present study, in order to assess changes in the bone marrow with aging, the fat tissue area, uncleated cell-count and cellularity in the bone marrow, in addition to changes in the diameter of the vascular lumen which result primarily from sclerotic changes in the dorsomedial artery of the bone marrow were determined in different age groups. The results revealed that all of the aforementioned factors changed significantly with aging. We also describe on the results of assays of inflammatory cytokines (IL-1, IL-6, TNF-alpha), lactoferrin and transferrin receptors in cases of anemia of chronic disorders (ACD) which own secondary to chronic inflammatory diseases and is known to frequently afflict the elderly.

Bone marrow failure: Pathophysiology and management

Morphologically, bone marrow is made up of a relatively mature but heterogenous population, fueled by a tiny pool of microscopically unrecognizable stem and progenitor cells. This complex tissue has the responsibility of maintaining our hematopoietic and, to a large extent, immunologic integrity, both of which are indispensable for health and, indeed, survival. Perhaps not surprisingly, bone marrow is the target of genetic, autoimmune, and environmental insults. Although robust, it has only a limited number of responses, one of which is reduction in cellular output, sometimes with superimposed qualitative abnormalities, and this is defined as bone marrow failure. Bone marrow failure is a diverse entity but can be logically explained and classified on a pathophysiologic basis. Thus the major recognizable categories of bone marrow failure are congenital and acquired defects. Each of these is subdivided according to the number of cell lines involved, over and above which the severity of the damage will determine reversibility. In each case, the natural history dictates management, and this ranges from short-term growth factor support to biologic immune response modulation and finally to bone marrow transplantation. In the past, many clinicopathologic variants of bone marrow failure were described, although their etiology was obscure and effective therapy was unavailable. This changed dramatically, however, when experimental hematologists, using radiobiology models, uncovered the dynamic nature of blood formation. Cardinal observations included the way in which spontaneous recovery followed irradiation, the central role played by pluripotential stem cells, and the integral participation of stroma in modulating this entire process. Understanding was refined once bone marrow cultures became available while, in parallel, the use of in-bred mouse strains launched the era of allogeneic transplantation. These approaches were combined, and the broad principles that govern basal or constitutive production emerged. Stem cells, with their characteristic commitment to self-renewal, exist at the apex of a hierarchy and generate a tier of proliferating progenitors that, in turn, give rise to a large postmitotic compartment of precursors that mature into distinctive myeloid and lymphoid lineages. The reserve potential is enormous, and output can be induced to meet even greatly increased demands. These events reflect the interaction of growth factors with a balancing set of negative regulators. The link between such diverse functions resides, to a large extent, in accessory cells and matrix geographically organized in what is now described as the hematopoietic inductive microenvironment. Many details of these meticulously orchestrated processes are obscure.(ABSTRACT TRUNCATED AT 400 WORDS)

Osteogenic Stem Cells and the Stromal System of Bone and Marrow

According to current hypothesis, cells of the osteogenic lineage, which includes both osteoblasts and chondroblasts, are derived from a stromal stem cell in the postnatal organism. That there exist osteogenic precursors in association with the soft, fibrous tissue of the marrow stroma is well established. An osteogenic tissue comprised of cartilage and bone is formed when marrow or marrow cell suspensions are cultured in vivo within diffusion chambers. Bone with a functional marrow organ is formed when marrow or marrow cell suspensions are transplanted heterotopically, e.g., under the renal capsule. Cultures of marrow stromal fibroblasts are readily established in vitro from single-cell bone marrow suspensions. Such cultures do not demonstrate overt differentiation in an osteogenic direction in vitro. When transplanted in vivo, however, they differentiate to form cartilage and bone in diffusion chambers and bone with a functional marrow organ when transplanted heterotopically. Single-cell bone marrow suspensions can be cultured in vitro under conditions that facilitate the formation of stromal fibroblast colonies. Circumstantial evidence supports the conclusion that each colony is derived from a single initiating cell termed a colony-forming unit-fibroblastic (CFU-F). A proportion of CFU-F demonstrates extensive proliferative potential both in vitro and in vivo. In vitro the extensive proliferative potential of a subset of CFU-F has been shown to be associated with a capacity for extensive self-renewal. On transplantation in vivo, the progeny of a proportion of CFU-F has been shown to be capable of proliferating and differentiating into all the stromal cell lines necessary for the formation of bone and reconstitution of the hematopoietic inductive microenvironment. These findings provide strong circumstantial evidence to support the hypothesis that there are stem cells present within the marrow stroma that are capable of giving rise to cells of a number of different lineages, including those of the osteogenic lineage (chondroblasts and osteoblasts).

Bone lining cells and hematopoiesis: an electron microscopic study of canine bone marrow.

Hematopoietic bone marrow in the dog is enclosed by a nearly complete and rather complex layer of endosteum, consisting of a diverse group of cells collectively called bone lining cells (BLC). Cell types comprising BLC include osteoblasts and osteoclasts, and other cell types, among which are elongated, flat cells with a spindle-shaped nucleus, and small cytoplasmic vesicles. The composition and thickness of the layer of BLC varies along the perimeter of the marrow. The layer may be simple or stratified. Occasionally a zone of tightly packed regularly arranged collagenous fibers lies between the bone lining cells and bone. Hematopoiesis, particularly neutrophilic, often occurs in the bone marrow next to the BLC. Cytoplasmic processes of BLC occasionally extend into the hematopoietic spaces and stromal cells in the hematopoietic compartment may extend processes to the layer of BLC. Occasionally cells of the BLC are similar in appearance to stromal cells within the marrow. Our observations together with the experimental findings of others (that fibroblastic stromal cells contribute to the hematopoietic inductive microenvironment, that hematopoietic stem cells are concentrated subosteally, that cells responsible for regeneration of the marrow stroma are derived from the endosteal layer, and that high concentrations of hematopoietic colony-stimulating factors are produced there) indicate that the hematopoietic capacities of bone marrow may be regulated by BLC.

Regeneration of murine megakaryocytopoiesis and the hematopoietic inductive microenvironment after sublethal whole body irradiation by treatment with an anabolic steroid.

The ability of the anabolic steroid nandrolone decanoate (ND) to increase megakaryocytopoiesis (MP) and the hematopoietic-inductive microenvironment (HIM) after sublethal irradiation was evaluated. Immediately after receiving whole body irradiation (2 Gy) and on the next 2 days, mice were injected with 1.25 mg of ND in propylene glycol intraperitoneally. Irradiated control mice received only vehicle. Compared to controls, drug-treated mice showed a 151% increase in the platelet level on day 14. Drug-treated mice also demonstrated a significant rebound in MP, as colony-forming progenitor stem cells (CFU-Mk) from bone marrow rose to 288% of control by day 8 and from spleen to 599% by day 1 following the last drug injection. In addition, drug-treated mice produced a 286% increase in bone marrow and a 378% in splenic stromal cell colonies on day 5 following drug treatment. These results demonstrate that ND enhances MP and HIM following sublethal irradiation. After hematopoietic suppression, ND may serve to stimulate MP and HIM recovery both at the stem cell level and in peripheral blood.

Effect of hydrocortisone and BCNU on long-term murine bone marrow cultures.

Hydrocortisone (HC) has been observed to influence an in vitro Dexter culture system with maintenance of pluripotent hematopoietic stem cells (CFU-s) and progenitor cells (CFU-gm, BFU-e). In the present study, a two-phase murine Dexter culture system was established to study the mechanism of HC-mediated suppression of stem cells or progenitor cells. Furthermore, the effect of exposing 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to an adherent cell layer for 1 h was investigated for maintenance of progenitor cells. Cultures supplemented with selenium and transferrin showed relatively good growth. The BFU-e number was significantly enhanced at 2 and 3 weeks of culture in the BCNU-treated group. This system is a good model for monitoring the influence of agents to a hematopoietic-inductive microenvironment.

Ability of lithium to accelerate the recovery of granulopoiesis after subacute radiation injury.

Lithium stimulates granulopoietic recovery after mice are exposed to 2 Gy. By examining the hematopoietic inductive microenvironment (HIM) using the stromal colony assay, we demonstrate here that lithium, during the two weeks after irradiation, produced less stromal colony suppression than was observed from the irradiated controls. Recovery peaked by day 19 and returned to normal by day 28. This response was also observed in splenic derived stroma. Furthermore, stroma from lithium-irradiated animals supported the in vitro growth of granulocyte-macrophage colonies (CFU-GM) greater than observed from irradiated controls. These data suggest lithium accelerates granulopoietic recovery by first providing for a completely reconstituted and functional HIM.

The regenerating liver: a site of erythropoiesis in the adult Long-Evans rat.

Erythropoiesis, which is primarily hepatic in the rat during fetal and early neonatal life, shifts almost entirely to the bone marrow in the neonatal-adolescent stage of development. In the adult, extramedullary erythropoiesis has been demonstrated in the liver and spleen under certain pathological conditions when bone marrow red cell production is insufficient. In the present study, erythropoietic foci have been found in young-adult rat liver regenerating 24-72 hr after subtotal hepatectomy. This erythropoiesis is both extravascular and sinusoidal, with some erythroblastic islands noted. The centrolobular hepatic area contains the highest concentration of erythroblasts. Peripheral blood reticulocytosis coincides with the appearance of these cells and this is considered as an indicator of effective erythropoiesis. Liver regenerating after partial hepatectomy produces significant quantities of erythropoietin (Ep) in response to hypoxia. Subtotal hepatectomy may confer upon the adult liver the ability to revert to a fetal-like condition both in its ability to produce Ep and to function as a hematopoietic inductive microenvironment for erythropoiesis.

Some observations of the hematopoietic status in vivo and in vitro on mice of genotype S1/S1d

Studies on the mechanism of anemia in mice of genotype S1/S1d have implicated the hematopoietic stroma (the hematopoietic inductive microenvironment, HIM) rather than hematopoietic stem cells as the site of the defect. Using methylcellulose-supported bone marrow culture systems, we have observed, in addition to classical hematopoietic colonies, the formation of surface associated fibroblastic plaques that could stimulate hematopoietic colony growth. These plaques were hypothesized to be derived from bone marrow stroma precursors. In view of the reported stromal-based defect in S1/S1d mice, studies were initiated, using our culture system, to determine if abnormalities exist in the plaque-forming potentials of these mice. Relative to controls, bone marrow derived from S1/S1d mice exhibited a significant decrease in hematopoietic colonly-forming units in culture, but no differences were apparent in the absolute numbers of fibroblastic plaque-forming units or in the ability of such plaques once derived to stimulate hematopoietic colony growth when overlain with fresh normal bone marrow preparations. Quantitative studies on the bone marrow of the S1/S1d mice revealed a marked reduction in total nucleated cells per femur. The importance of evaluating the results of bone marrow cultures in an absolute (i.e., number of units per femur) rather than a relative (i.e., number of units forming in a constant cell inoculum) term was underlined by these studies.

Some Observations of the Hematopoietic Status In Vivo and In Vitro on Mice of Genotype S1/S1d

Abstract Studies on the mechanism of anemia in mice of genotype S1/S1d have implicated the hematopoietic stroma (the hematopoietic inductive microenvironment, HIM) rather than hematopoietic stem cells as the site of the defect. Using methylcellulose-supported bone marrow culture systems, we have observed, in addition to classical hematopoietic colonies, the formation of surface associated fibroblastic plaques that could stimulate hematopoietic colony growth. These plaques were hypothesized to be derived from bone marrow stroma precursors. In view of the reported stromal-based defect in S1/S1d mice, studies were initiated, using our culture system, to determine if abnormalities exist in the plaque-forming potentials of these mice. Relative to controls, bone marrow derived from S1/S1d mice exhibited a significant decrease in hematopoietic colonly-forming units in culture, but no differences were apparent in the absolute numbers of fibroblastic plaque-forming units or in the ability of such plaques once derived to stimulate hematopoietic colony growth when overlain with fresh normal bone marrow preparations. Quantitative studies on the bone marrow of the S1/S1d mice revealed a marked reduction in total nucleated cells per femur. The importance of evaluating the results of bone marrow cultures in an absolute (i.e., number of units per femur) rather than a relative (i.e., number of units forming in a constant cell inoculum) term was underlined by these studies.