Review: tissue engineering: reconstitution of human hematopoiesis ex vivo

The reconstruction of functioning human tissues ex vivo is becoming an important part of biotechnology. There are compelling scientific, clinical, and biotechnological reasons for fully or partially reconstituting human tissues such as skin, bone marrow, and liver ex vivo. In particular, bone marrow is a tissue of much importance, and there are significant societal and health benefits derived from a successfully constructed ex vivo hematopoietic system. In this article, we review the current status of this effort. The topics covered include the current understanding of the biology of human hematopoiesis, the motivation for reconstructing it ex vivo, the current state of ex vivo human hematopoietic cultures, the development of important metrics to judge culture performance, and an approach based on in vivo mimetics to accomplish this goal. We discuss some applications of functional ex vivo hematopoietic cultures and the biological and engineering challenges that face research in this area. (c) 1993 John Wiley & Sons, Inc.     

Young donor hematopoietic stem cells revitalize aged or damaged bone marrow niche by transdifferentiating into functional niche cells

The bone marrow niche maintains hematopoietic stem cell (HSC) homeostasis and declines in function in the physiologically aging population and in patients with hematological malignancies. A fundamental question is now whether and how HSCs are able to renew or repair their niche. Here, we show that disabling HSCs based on disrupting autophagy accelerated niche aging in mice, whereas transplantation of young, but not aged or impaired, donor HSCs normalized niche cell populations and restored niche factors in host mice carrying an artificially harassed niche and in physiologically aged host mice, as well as in leukemia patients. Mechanistically, HSCs, identified using a donor lineage fluorescence-tracing system, transdifferentiate in an autophagy-dependent manner into functional niche cells in the host that include mesenchymal stromal cells and endothelial cells, previously regarded as “nonhematopoietic” sources. Our findings thus identify young donor HSCs as a primary parental source of the niche, thereby suggesting a clinical solution to revitalizing aged or damaged bone marrow hematopoietic niche.     

The influence of extra-cellular matrix and stroma remodeling on the productivity of long-term human bone marrow cultures

The stromal cell layer is believed to play an important role in long-term human bone marrow cultures (LTHBMCs). At present, neither the role that the stromal cell extra-cellular matrix (ECM) plays in influencing stroma behavior is well understood nor are the effects of stroma aging. Rapid medium exchanged LTHBMCs were established on surfaces precoated with human natural fibronectin and type 1 rat tail collagen. Although initial adhesion of hematopoietic cells was improved by the presence of both ECMs, the overall progenitor and nonadherent cell productivity was not improved nor did the stroma grow to confluency faster. Thus, the ECMs used did not significantly influence the cell productivity of LTHBMCs. To examine the influence of stromal cell layer aging, conditioned medium was obtained from the first two weeks of LTHBMCs that was subsequently concentrated and used as a medium supplement in a second set of slowly exchanged LTHBMCs. The presence of the concentrated conditioned medium (conCM) enhanced the production of nonadherent cells three-fold compared with control over an eight week culture period. Control cultures that were exposed to conCM after 4 weeks in culture significantly improved their cell productivity during the latter 4 weeks of culture compared with control. The productivity of cultures exposed to conCM for 4 weeks dropped significantly when unsupplemented medium was used for the latter 4 weeks of culture. Interestingly, phytohemagglutin-stimulated leukocyte-conditioned medium stimulated LTHMBCs in a similar fashion, as did conditioned medium from early LTHBMCs. Taken together, these results strongly suggest that the stromal cell layer does produce important factors for active hematopoiesis during its growth to confluence.     

Uncovering the emergence of HSCs in the human fetal bone marrow by single-cell RNA-seq analysis

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Frequent harvesting from perfused bone marrow cultures results in increased overall cell and progenitor expansion

The establishment of prolific long-term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10- to 30-fold increase in total cell number and in the number of colony forming units-granulocyte/macrophage (CFU-GMs), and a four- to tenfold increase in the number of long-term culture initiating cells (LTC-ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading to stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion-based bioreactor systems. One possible constraint is the size of the growth surface area required. This constraint can be overcome by harvesting half the cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100-fold and the CFU-GM expansion exceeded 30-fold over a 27-day period. These calculated values are based on growth that could be obtained from the harvested cell population. Growth of the adherent cell layer was stable, whereas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols published to date are suboptimal for long-term cultivation, and that further definition and refinement is likely to lead to even greater expansion of hematopoietic cell populations obtained from bone marrow. More importantly, these results show that the LTC-IC measured during the single pass expansion do have further expansion potential that can be realized by frequent harvesting. Finally, the present culture conditions provide a basis for an assay system for the identifications provide a basis for an assay system for the identification of the factors that determine the long-term maintenance and replication of human stem cells ex vivo. (c) 1994 John Wiley & Sons, Inc.     

胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

Decline in IGF1 in the bone marrow microenvironment initiates hematopoietic stem cell aging

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Oncostatin M regulates mesenchymal cell differentiation and enhances hematopoietic supportive activity of bone marrow stromal cell lines

Bone marrow stromal cell lines (TBR cell lines) established from temperature-sensitive Simian Virus 40 T-antigen gene transgenic mice exhibited myogenic, osteogenic, and adipogenic differentiation. The effect of oncostatin M (OSM) on such mesenchymal cell differentiation of marrow stromal cell lines was examined. One of those stromal cell lines, TBRB, differentiated into skeletal muscle, and its differentiation was stimulated by OSM, whereas differentiation of TBR10-1 into smooth muscle was inhibited by OSM. TBR31-2 is a bipotent progenitor for adipocytes and osteoblasts, and OSM stimulated osteogenic differentiation while inhibiting adipogenic differentiation. On the other hand, TBR cell lines exhibited various potentials for supporting hematopoiesis in culture. When hematopoietic progenitor cells were cocultured with OSM-stimulated stromal cell lines, TBR10-1 and TBR31-2 exhibited enhanced hematopoietic supportive activity. As responsible molecules for stromal cell dependent hematopoiesis, expression of stem cell factor (SCF) (a ligand of c-Kit), vascular cell adhesion molecule (VCAM-1) (a ligand of VLA-4), and secretion of interleukin (IL)-6 were increased by OSM. OSM affected mesenchymal cell differentiation and promoted the hematopoietic supportive activity of marrow stromal cell lines. As OSM production is induced by cytokines from hematopoietic cells, OSM may be a key factor in mutual regulation between hematopoietic cells and stromal cells in the bone marrow. OSM may play a role as a regulator in maintaining the hematopoietic microenvironment in marrow by coordinating mesenchymal differentiation.     

Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates

Background

The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning.

Methods

Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34⁺ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells.

Results

Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes.

Conclusions

Low levels of GFP‐transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

In vivo evaluation of bioprinted prevascularized bone tissue

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.     

The microbiota regulates hematopoietic stem cell fate decisions by controlling iron availability in bone marrow

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Human hematopoietic stem cell vulnerability to ferroptosis

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Large-scale production and directed induction of functional dendritic cells ex vivo from serum-free expanded human hematopoietic stem cells

BackgroundDendritic cells (DCs) that are derived from hematopoietic stem cells (HSCs) are the most potent antigen-presenting cells and play a pivotal role in initiating the immune response. Hence, large-scale production and direct induction of functional DCs ex vivo from HSCs are crucial to HSC research and clinical potential, such as vaccines for cancer and immune therapy.MethodsIn a previous study, we developed a serum-free HSC expansion system (SF-HSC medium) to expand large numbers of primitive HSCs ex vivo. Herein, a DC induction and expansion medium (DC medium) was proposed to further generate large numbers of functional DCs from serum-free expanded HSCs, which were developed and optimized by factorial design and the steepest ascent method.ResultsThe DC medium is composed of effective basal medium (Iscove's modified Dulbecco's medium [IMDM]) and cytokines (2.9 ng/mL stem cell factor [SCF], 2.1 ng/mL Flt-3 ligand, 3.6 ng/mL interleukin [IL]-1β, 19.3 ng/mL granulocyte-macrophage colony-stimulating factor [GM-CSF] and 20.0 ng/mL tumor necrosis factor-α [TNF-α]). After 10-day culture in DC medium, the maximum fold expansion for accumulated CD1a⁺CD11c⁺ DCs was more than 4000-fold, and the induced DCs were characterized and confirmed by analysis of growth kinetics, surface antigen expression, endocytosis ability, mixed lymphocyte reaction, specific cytokine secretion and lipopolysaccharide stimulation.DiscussionIn conclusion, the combination of DC medium and SF-HSC medium can efficiently induce and expand a large amount of functional DCs from a small scale of HSCs and might be a promising source of DCs for vaccine and immune therapy in the near future.     

Regulation of hematopoiesis and the hematopoietic stem cell niche by Wnt signaling pathways

Hematopoietic stem cells （HSCs） are a rare population of cells that are responsible for life-long generation of blood cells of all lineages. In order to maintain their numbers, HSCs must establish a balance between the opposing cell fates of self-renewal （in which the ability to function as HSCs is retained） and initiation of hematopoietic differentiation. Multiple signaling pathways have been implicated in the regulation of HSC cell fate. One such set of pathways are those activated by the Wnt family of ligands. Wnt signaling pathways play a crucial role during embryogenesis and deregulation of these pathways has been implicated in the formation of solid tumors. Wnt signaling also plays a role in the regulation of stem cells from multiple tissues, such as embryonic, epidermal, and intestinal stem cells. However, the function of Wnt signaling in HSC biology is still controversial. In this review, we will discuss the basic characteristics of the adult HSC and its regulatory microenvironment, the ＂niche＂, focusing on the regulation of the HSC and its niche by the Wnt signaling pathways.     

Kinetic Analysis of the ex vivo Expansion of Human Hematopoietic Stem/Progenitor Cells

The total cell expansion of human umbilical cord blood (CB) and adult bone marrow (BM) CD34+-enriched cells cultured in supplemented serum-free media, either over irradiated human feeder layers or in stroma-free systems, were characterized by a simple kinetic model using only two parameters: the specific cell expansion rate, mu, and the death rate constant, k(k). Both CB and BM cells can expand at approximately the same rate (0.21 day(-1)) in this culture system however, cell death depends on the presence of stroma and the environment in which the cells are cultured.     

Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.