Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear‐cut effects on cell proliferation,differentiation and metabolism

Physiologically low O₂ tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0–5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O₂ tensions (3–10%). We hypothesized that O₂ concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34⁺‐enriched cells was studied in co‐culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O₂. 2% O₂ resulted in a significantly lower CD34⁺ cell expansion (25‐fold vs 60‐, 64‐ and 92‐fold at day 10 for 5%, 21%, 10% O₂, respectively). In turn, 10% O₂ promoted the highest CD34⁺CD90⁺ cell expansion, reaching 22 ± 5.4‐ vs 5.6 ± 2.4‐ and 5.7 ± 2.0‐fold for 2%, 5% and 21% O₂, respectively, after 14 days. Similar differentiation patterns were observed under different O₂ tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O₂. Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O₂ when compared to higher O₂ concentrations (5–21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O₂, can maximize the ex vivo expansion of UCB stem/progenitor cells in co‐culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

New approaches to expand hematopoietic stem and progenitor cells

Introduction: Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and to differentiate into all blood cell lineages, and are currently the foundation of HSC transplantation therapy. A variety of methods have recently been explored to find a way to expand hematopoietic stem and progenitor cells (HSCs/PCs) ex vivo in order to improve the efficiency and outcome of HSC transplantation.

Areas covered: Recent studies of HSCs/PCs have led to the development of new ways to detect and purify HSCs/PCs and have also revealed several intrinsic and extrinsic factors that control the molecular signals fundamental to self-renewal and differentiation of HSCs. These findings have provided new approaches for expanding HSCs/PCs ex vivo utilizing protein factors and small-molecule compounds (SMCs) and have also demonstrated promising outcomes in clinical trials.

Expert opinion: Although further technical innovation is still needed, elucidation of the whole picture of signaling pathways critical to HSCs/PCs and manipulation of such pathways by SMCs could establish efficient, cost-effective, riskless and robust methods for ex vivo expansion of HSCs/PCs. With these efforts, more sophisticated HSC transplantation would be possible in the near future.     

骨髓间充质干细胞分化的成骨细胞支持脐血造血干祖细胞的研究

本研究利用骨髓间充质干细胞诱导分化的成骨细胞构建二维培养体系,探讨其体外支持脐血干/祖细胞生存的作用.体外进行人骨髓间充质干细胞（MSC）原代培养后诱导为成骨细胞,构建二维培养体系.将免疫磁珠分选的CD34^＋脐血干/祖细胞接种于其中,在无外源性细胞因子情况下进行体外培养,进行CFU,HPP-CFU和LTC-IC测定并将其分别与MSC、成骨细胞,MSC/成骨细胞（1：2）作为饲养细胞的二维培养体系实验结果相比较.结果发现：在所构建的造血干/祖细胞（HSPC）二维培养体系中,骨髓MSC诱导成骨细胞对于脐血造血干/祖细胞培养的支持作用显著优于其他各组培养体系,尤其对长期造血干细胞（long term-HSC）体外生存有更为明显地支持作用.结论：骨髓间充质干细胞诱导成骨细胞构建的二维体系对于脐血干/祖细胞体外培养具有支持作用,进一步证实成骨细胞对造血干细胞生存与增殖具有重要调控作用.     

Ex vivo expansion of cord blood‐CD34+ cells using IGFBP2 and Angptl‐5 impairs short‐term lymphoid repopulation in vivo

Cord blood‐derived haematopoietic stem cells (CB‐HSCs) are an attractive source for transplantation in haematopoietic disorders. However, the yield of CB‐HSCs per graft is limited and often insufficient, particularly for the treatment of adult patients. Here we compare the capacity of three cytokine cocktails to expand CB‐CD34⁺ cells. Cells were cultured for 5 or 14 days in media supplemented with: (a) SCF, FL, IL‐3 and IL‐6 (SFLIL3/6); (b) SCF, TPO, FGF‐1 and IL‐6 (STFIL6); and (c) SCF, TPO, FGF‐1, IGFBP₂ and Angptl‐5 (STFAI). We observed that STFAI‐culture expansion sustained the most vigorous cell proliferation, maintenance of CD34⁺ phenotype and colony‐forming unit counts. In addition, STFAI‐cultured cells had a potent ex vivo migration activity. STFAI‐expanded cells were able to engraft NSG mice. However, no significant difference in overall engraftment was observed among the expansion cocktails. Assessment of short‐term reconstitution using multilineage markers demonstrated that the STFAI cocktail for HSCs expansion greatly improved total cell expansion but may impair short‐term lymphoid repopulation. Copyright © 2012 John Wiley & Sons, Ltd.     

Carlecortemcel-l: an ex vivo expanded umbilical cord blood cell graft for allogeneic transplantation

Background: Success of umbilical cord blood transplantation (UCBT) is mostly affected by the cell dose infused and its application is limited by the size of the recipient. For most adults and older children it is not possible to find a single UCB unit large enough for reliable engraftment. One strategy to increase the number of progenitor cells available is ex vivo expansion of the unit. The main challenge of ex vivo expansion systems is how not to deplete the self-renewing cell population by driving them into differentiation into committed progenitors. Objective: Copper modulates basic cell functions, such as survival, proliferation, and differentiation. Reduction of cellular copper in ex vivo culture conditions enabled preferential proliferation of early progenitors and increased engraftment capabilities. The result of a Phase I study of carlecortemcel-l, a product derived from ex vivo expansion of UCB progenitors in the presence of a copper chelator and early-acting cytokines, and the study design for the current pivotal study are presented. Methods: A literature review using PubMed and the investigator's brochure from the manufacturer. Conclusions: Early results suggest that carlecortemcel-l infusion is safe and may be associated with favorable non-relapse mortality rates. A pivotal global study is currently being conducted to evaluate safety and efficacy of this product from centralized manufacturing facilities.     

Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry

Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so‐called ‘haematopoietic niche’. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long‐term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum‐free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280‐fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion. Copyright © 2012 John Wiley & Sons, Ltd.     

New approaches to expand hematopoietic stem and progenitor cells

INTRODUCTION: Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and to differentiate into all blood cell lineages, and are currently the foundation of HSC transplantation therapy. A variety of methods have recently been explored to find a way to expand hematopoietic stem and progenitor cells (HSCs/PCs) ex vivo in order to improve the efficiency and outcome of HSC transplantation. AREAS COVERED: Recent studies of HSCs/PCs have led to the development of new ways to detect and purify HSCs/PCs and have also revealed several intrinsic and extrinsic factors that control the molecular signals fundamental to self-renewal and differentiation of HSCs. These findings have provided new approaches for expanding HSCs/PCs ex vivo utilizing protein factors and small-molecule compounds (SMCs) and have also demonstrated promising outcomes in clinical trials. EXPERT OPINION: Although further technical innovation is still needed, elucidation of the whole picture of signaling pathways critical to HSCs/PCs and manipulation of such pathways by SMCs could establish efficient, cost-effective, riskless and robust methods for ex vivo expansion of HSCs/PCs. With these efforts, more sophisticated HSC transplantation would be possible in the near future.     

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.     

Controlling culture dynamics for the expansion of hematopoietic stem cells

The ex vivo expansion of hematopoietic stem cells (HSCs) is the subject of intense commercial and academic interest due to the potential of HSCs to be a renewable source of material for cellular therapeutics. Unfortunately, because methodologies have not yet been developed to grow clinically relevant numbers of HSCs (or their derivatives) consistently, the potential of this technology is limited. Manipulation of the in vitro culture microenvironment, primarily through cytokine supplementation, has been the predominant approach in studies attempting to expand primary human HSC numbers in vitro. While promising results have been obtained, it is becoming clear that novel methods must be developed before cellular therapies using these stem cells can become routine. Ideally, bioprocesses must be designed to target specifically the growth of stem cell populations while incorporating positive and negative feedback from potentially dynamic mature and maturing cell populations. The product of these culture systems should consist of not only HSCs, but also of cells that allow the engraftment of HSCs and, ideally, cells responsible for the immediate or accelerated functional support of patients. Development of such "designer transplants" will require combining optimal culture conditions capable of amplifying HSC numbers with novel approaches for finely controlling the number, functional capabilities, and characteristics of potentially therapeutic cells in these very complex cell culture systems.     

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS‐5 stromal cell line

Lineage‐specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bone marrow failure and immunodeficiencies. The current methods for HSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell‐free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell‐free matrices from a murine bone marrow stromal cell line (MS‐5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O₂ tensions. These acellular matrices were used as biological scaffolds for the lineage‐specific expansion of magnetically sorted CD34⁺ cells and the results were evaluated by flow cytometry and colony‐forming assays. We could get up to 80‐fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering‐based ex vivo HSPC expansion, and it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow. Copyright © 2012 John Wiley & Sons, Ltd.     

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.     

Fucose‐deficient hematopoietic stem cells have decreased self‐renewal and aberrant marrow niche occupancy

BACKGROUND: Modification of Notch receptors by O‐linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study was to assess the self‐renewal and stem cell niche features of fucose‐deficient HSCs. STUDY DESIGN AND METHODS: Homeostasis and maintenance of HSCs derived from FX^?/? mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two‐photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose‐deficient and wild‐type (WT) HSCs. RESULTS: Marrow progenitors from FX^?/? mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose‐deficient HSCs from FX^?/? mice displayed decreased self‐renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by two‐photon intravital imaging, the fucose‐deficient HSCs were found localized farther from the endosteum of the calvarium marrow than the WT HSCs. CONCLUSIONS: The current reported aberrant niche occupancy by HSCs from FX^?/? mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O‐fucosylation, suggests that fucosylation‐modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.     

Establishment of a cell processing laboratory to support hematopoietic stem cell transplantation and chimeric antigen receptor (CAR)-T cell therapy

Cell processing laboratories are an important part of cancer treatment centers. Cell processing laboratories began by supporting hematopoietic stem cell (HSC) transplantation programs. These laboratories adapted closed bag systems, centrifuges, sterile connecting devices and other equipment used in transfusion services/blood banks to remove red blood cells and plasma from marrow and peripheral blood stem cells products. The success of cellular cancer immunotherapies such as Chimeric Antigen Receptor (CAR) T-cells has increased the importance of cell processing laboratories. Since many of the diseases successfully treated by CAR T-cell therapy are also treated by HSC transplantation and since HSC transplantation teams are well suited to manage patients treated with CAR T-cells, many cell processing laboratories have begun to produce CAR T-cells. The methods that have been used to process HSCs have been modified for T-cell enrichment, culture, stimulation, transduction and expansion for CAR T-cell production. While processing laboratories are well suited to manufacture CAR T-cells and other cellular therapies, producing these therapies is challenging. The manufacture of cellular therapies requires specialized facilities which are costly to build and maintain. The supplies and reagents, especially vectors, can also be expensive. Finally, highly skilled staff are required. The use of automated equipment for cell production may reduce labor requirements and the cost of facilities. The steps used to produce CAR T-cells are reviewed, as well as various strategies for establishing a laboratory to manufacture these cells.     

Intersecting Worlds of Transfusion and Transplantation Medicine: An International Symposium Organized by the Canadian Blood Services Centre for Innovation

The principal theme of the symposium was centered on how the world of regenerative medicine intersects with that of transfusion medicine, with a particular focus on hematopoietic stem cells (HSCs) and stem cell therapies. The symposium highlighted several exciting developments and identified areas where additional research is needed. A revised map of human hematopoietic hierarchy was presented based on the functional and phenotypic analysis of thousands of single stem and progenitor cells from adult bone marrow and fetal liver. These analyses revealed that multipotency is largely restricted to the HSC and multipotent progenitor compartments in adult bone marrow where most progenitors are unipotent, whereas fetal liver contains a large number of distinct oligopotent progenitors. Furthermore, unlike adult bone marrow, multipotency is extended in the downstream progenitors in the hierarchy in the fetal liver stage. Production of platelets ex vivo from HSCs is emerging as a potentially viable option because of advances in culture techniques that combine cytokine mixtures, small molecules, and shear stress. However, limited HSC expansion and low platelet yield from culture-derived megakaryocytes remain problematic. Evidence was presented to support stricter guidelines for transfusion of platelets and red blood cells practices in allogeneic HSC transplant patients, although evidence is often extrapolated from general indications. Basic principles of human leukocyte antigen testing in HSC transplant were described, emphasizing the need for a national (and global) stem cell donor registry. Ongoing research is aimed at improving cellular cryopreservation including the establishment of a new thawing protocol that improves viability of umbilical cord blood CD34+ cells. Umbilical cord blood transplantation practices have also been improved; recent studies suggest noninferior outcomes when patients are transplanted with umbilical cord blood vs a matched adult donor. Finally, mesenchymal stem cell infusion is an example of a cellular therapy useful for immunomodulation. Preclinical trials suggest that mesenchymal stem cells may be effective in managing sepsis. In conclusion, practices and research surrounding HSCs are continuing to evolve rapidly as new information is obtained.     

Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells

Hematopoietic stem cell (HSC) populations change with aging, but the extent to which this is caused by qualitative versus quantitative alterations in HSC subtypes is unclear. Using clonal assays, in this study we show that the aging HSC compartment undergoes both quantitative and qualitative changes. We observed a variable increase of HSC pool size with age, accompanied by the accumulation of predominantly myeloid-biased HSCs that regenerate substantially fewer mature progeny than young myeloid-biased HSCs and exhibit reduced self-renewal activity as measured by long-term secondary transplantation. Old HSCs had a twofold reduction in marrow-homing efficiency and a similar decrease in functional frequency as measured using long-term transplantation assays. Similarly, old HSCs had a twofold reduced seeding efficiency and a significantly delayed proliferative response compared with young HSCs in long-term stromal cell co-cultures but were indistinguishable in suspension cultures. We show that these functional defects are characteristics of most or all old HSCs and are not indicative of a nonfunctional subset of cells that express HSC markers. Furthermore, we demonstrate that cells with functional properties of old HSCs can be generated directly from young HSCs by extended serial transplantation, which is consistent with the possibility that they arise through a process of cellular aging.     

Mesenchymal stem cells from CD34− human umbilical cord blood

Umbilical cord blood (UCB) is well known to be a rich source of stem cells especially for haematopoietic stem cells (HSCs). Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. Although MSCs have been described by a subset of surface antigens after expansion, little is known about the cell surface phenotype of undifferentiated MSCs. The aim of this study therefore was to clarify whether undifferentiated MSCs are resident among CD34^? UCB cells. CD34⁺ cells were separated from UCB mononuclear cells (MNCs) by magnetic sorting and the CD34^? cell fractions were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and basic‐fibroblast growth factor. Isolated CD34⁺ cells were also cultured in the same medium. Adherent fibroblast‐like cells at passage 3–4 were analyzed by fluorescence‐activated cell sorting (FACS) for MSC marker expression , and standard adipogenic, osteogenic and chondrogenic assays were used to investigate their differentiation potentials. After 4–5 weeks in culture, the cells from the CD34^? fraction became confluent with flat and fibroblast‐like morphology. These cells were positively stained for the mesenchymal cell markers CD29, CD73 and CD105. In adipogenic differentiation, the cells showed oil red O positive and expressed FABP4, adipsin and proliferation‐activated receptor γ‐2 (PPARγ2 genes) associated with adipogenesis. In osteogenic differentiation, calcium accumulation and osteocalcin were detected. The cells grown in chondrogenic conditions were positively stained for human aggrecan and expressed collagen type II and Sox‐9 genes. In contrast, cells from the CD34⁺ fraction failed to generate any cells with MSC morphology under the same culture conditions. Our results showed that UCB contained MSCs which are only resident in the CD34^? fraction. The MSCs could be induced to differentiate into at least three lineage cell types, adipocytes, osteoblasts and chondrocytes.     

Hematopoietic stem cell gene therapy: selecting only the best

Hematopoietic stem cell (HSC) gene therapy can potentially cure a variety of human hematopoietic diseases, such as sickle cell disease. Selection and expansion of gene-corrected HSCs has now been accomplished for the first time using HSC from large animals - dogs and humans - with a novel drug-resistance gene, MGMT, which is not expressed in normal HSCs (see the related articles beginning on pages 1561 and 1581). Highly efficient lentiviral transfer and expression of MGMT into relatively few HSCs led to repopulation of most of the hematopoietic compartment with gene-corrected cells following suitable drug treatment. This selection system may be useful in human clinical trials to permit gene therapy in autologous and allogeneic bone marrow transplantation settings.