Umbilical cord blood stem cells: Towards a proteomic approach

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / j p r o t

Review

Umbilical cord blood stem cells: Towards a proteomic approach Angelo D'Alessandroa,b , Giancarlo Liumbrunob,c , Giuliano Grazzinib , Simonetta Pupellab , Letizia Lombardinid , Lello Zollaa,⁎ a

Department of Environmental Sciences, Tuscia University, Viterbo, Italy Italian National Blood Centre, Istituto Superiore di Sanità, Rome, Italy c Immunohematology and Transfusion Medicine Unit, “San Giovanni Calibita” Fatebenefratelli Hospital, Rome, Italy d Italian National Transplant Centre, Istituto Superiore di Sanità, Rome, Italy b

AR TIC LE D ATA

ABSTR ACT

Keywords:

The first umbilical cord blood (UCB) transplant to a sibling with Fanconi's anaemia in 1988

Cord blood

represented a breakthrough in the field of transplantation. Thereon, several transplants

Stem cells

have been performed with UCB-derived hematopoietic stem cells (HSCs) and a plethora of

Proteomics

studies have investigated the plasticity of UCB-derived stem and progenitor cells. However,

CD34

these studies have not been hitherto translated into clinical trials and, although UCB is

Mass spectrometry

routinely used as an alternative source of HSCs, no substantial advances have been made in

Transfusion medicine

the field of clinical regenerative medicine. The real deal is the lack of knowledge about the molecular processes governing the events of differentiation which transform immature UCB stem cells into terminally-committed hematopoietic, muscle, bone and nervous cells. In order to fill this void, several studies have been recently focused on the identification of the peculiar proteomic profile of UCB-derived stem cells. Hereby, we concisely review recent proteomic surveys addressing UCB-derived stem and progenitor cells. Notably, comparative studies detected a wider spectrum of proteins in immature cells rather than in more differentiated populations, as if maturation events could represent a bottleneck to protein expression. Future research projects should try to shed light on these processes and their completion could pave the way for unprecedented treatments. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . 1.1. UCB: therapeutic stem cells. UCB and proteomics. . . . . . . . . 2.1. Why a proteomic approach .

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Abbreviations: ASC, adult stem cell; CAFC, cobblestone area forming cell; CD, cluster designation; ESC, embryonic stem cell; FGF, fibroblast growth factor; GM-CSF, granulocyte monocyte colony stimulating factor; GVHD, Graft versus host disease; GVL, graft versus leukaemia; HLA, human leukocyte antigen; HSC, hematopoietic stem cell; IL, interleukin; LTC-IC, long term-culture initiating cell; MSC, mesenchymal stem cell; NOD/SCID, non-obese diabetic/severe combined immunodeficient; SCF, stem cell factor; S/PC, stem and progenitor cell; TPO, thrombopoietin; UCB, umbilical cord blood; VLA, very large antigen; XIC, extracted ion current. ⁎ Corresponding author. E-mail address: [email protected] (L. Zolla). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.06.009

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2.2. CD34+: a suitable marker for targeted proteomics . . . . . . . 2.3. MSCs and cellular therapeutics: recent proteomics advances 3. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

Introduction

More than fifty years have already passed since E.D. Thomas carried out the first bone marrow transplant in 1957 [1]. Forthwith, the transplantation treatment has turned out to be a lifesaving approach for a growing number of pathologies (Table 1). Indeed, hematopoietic stem cells (HSCs), from bone marrow or peripheral blood, currently represent a therapeutic largely-exploited resource in the transplantation endeavour [2,3]. Nonetheless, until recently the likelihood of performing a transplant was strictly tied to the availability of a suitable donor who was either totally compatible with the patient or a close family member. Even in the latter case, the odds were discouraging (for example, only the 25% of the siblings usually matched compatibility requirements). The continuous refinement of the transplantation techniques has progressively widened the applicability range for these procedures. In parallel, in order to face the growing demand for suitable donors, the institution of international registries has progressively become an impelling exigency

Table 1 – Diseases treated by cord blood transplantation. Acute lymphocytic leukaemia Acute myeloid leukaemia Adrenoleukodystrophy Amegakaryocytic thrombocytopenia Blackfan–Diamond syndrome Bone marrow aplasia Chronic lymphocytic leukaemia Chronic myeloid leukaemia Dyskeratosis congenital Fanconi's anaemia Globoid cell leukodystrophy Gunther disease Hodgkin's and non-Hodgkin's lymphoma Hunter syndrome Hurler syndrome Idiopathic aplastic anaemia Kostmann syndrome Lesch–Nyhan syndrome Multiple myeloma Myelodysplastic syndrome Myelofibrosis with myeloid metaplasia Neuroblastoma Osteopetrosis Primitive immune deficiencies Severe combined immune deficiency Sickle cell anaemia Soft tissue Sarcoma Thalassaemia Wiskott–Aldrich syndrome X-linked lymphoproliferative syndrome Data collected from [3,17,76]

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and, soon afterwards, a solid reality with over 12 million “virtual” voluntary donors actually scheduled. Nevertheless, the search for a matching donor still remains a long-lasting quest (from 4 to 6 months), whereas most of the patients unfortunately do not have such a long life expectancy. Furthermore, this search has elevated costs and the risk of donor cancellation, either voluntary or due to clinical causes, should be taken into account in approximately 1% of the cases [4]. Therefore, it is easy to understand why the clinical milieu has begun to wonder whether it would be possible to base on alternative HSC sources. In this respect, umbilical cord blood (UCB) has been attracting a great deal of interest since the paper by Knudtzon et al. was published in 1974 [5], whereby evidences were provided of the presence of hematopoietic colony-forming cells in human UCB. However, the first thorough study in this field dates back to 1982, when discussions between Broxmeyer and Boyse led to a series of experiments that undoubtedly individuated HSCs in the UCB [6]. Owing to these results, a possible role was suggested for UCB-derived HSCs in bone marrow replacement [7]. It was only in 1988 that the first successful cord cell transplant to a sibling with Fanconi's anaemia took place [4]. Soon afterwards, in 1989, Harris and colleagues performed the first matched sibling cord blood transplant in the United States [8]. A few years later, the question was raised whether UCB could be used for transplantation from an unrelated donor. A positive answer was given by Kurtzberg et al. in 1993 [9]. Finally, it became fairly evident that UCB could be exploited as an alternative source of HSCs [10], although a deeper analysis was necessary on the outcome of UCB related- or unrelatedtransplants. A plethora of authors gave their contribution to this specific cause [11–15]. Actually, UCB represents a biological fruitful element – albeit it was previously considered as “a waste product” – and prevents the donor to suffer from all the problems related to the painful and invasive surgery procedures hampering bone marrow donations. The biological characteristics of UCB also allow to overcome the traditional compatibility barriers, since it is possible to perform an UCB-HSC transplant also between non-perfectly compatible subjects, with a reduction in the number of complications, such as rejection and acute or chronic graft versus host disease (GVHD) [16]. The possibility to perform transplants of HSCs from UCB has led to the creation of “real” – no more “virtual” – banks, where the collected UCB units are actually stored [17,18]. The number of UCB banks has dramatically increased in the last few years [19]. In parallel, the World Marrow Donor Association has recently reported a net increase in the number of donations of stem cells for unrelated patients during the last decade, especially from alternative sources such as peripheral and cord blood (Fig. 1) [19]. The boosted use of UCB as an alternative source of HSCs (Fig. 1) has enabled physicians to further feedback the growing

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Fig. 1 – World Marrow Donor Association has been registering a drastic increase in stem cell products provided for unrelated patients [64]. While bone marrow donations have quite stabilized in the last decade, alternative sources of HSC have been individuated in peripheral blood and umbilical cord blood, whose donation rose exponentially in the last 5 years. Adapted from Brand et al. [19].

transplantation request [20,21]. Indeed, over 400,000 samples have been collected and frozen [22–29] worldwide and are currently available for transplantation [19] (Table 2). The benefits – both organizational and biological – related to the use of HSCs from UCB are the following: ease of availability, faster donor identification, the possibility to find donors for patients from ethnic minorities (which are conversely underrepresented in marrow donor registries), the ability to perform transplants even if donors and recipients are not fully compatible, the lower severity of immunological complications such as GVHD and the reduced risk of transmission of infectious diseases [30–32]. Notwithstanding, some concerns dampen the enthusiasm around UCB as the principal source of HSCs. To begin with, the volumes that can be collected from a single donor of UCB and bone marrow are not equivalent. Moreover, despite the high concentration of UCB hematopoietic stem and progenitor cells (S/PCs) and their increased proliferative capacity, the absolute number of cells within a single unit is very low when compared

to bone marrow sources (1–3% of bone marrow versus 0.5–1% of UCB). Therefore, it frequently happens that the UCB nucleated cell dose does not reach the lower acceptable limit for an adult, which is approximately 1.5–2 × 107/kg body weight [17]. Thus, recovery after UCB-HSC transplantation is sensibly delayed since post-transplant hematopoietic reconstitution period is proportional to the original infused cell dose and, therefore, significantly longer. This has meant that, for a long time, the use of UCB-HSCs has been almost exclusively limited to the treatment of paediatric patients. However, recent experiences have shown promising results in adult patients as well [33–38]. In order to cope with the low-cell dose/unit related-problems, new techniques of expansion ex vivo [39–42], non myeloablative conditioning regimens [43], intra-bone inoculation of UCB [44] and multiple-unit infusions were introduced [45–47].

1.1.

UCB: therapeutic stem cells

Stem cells could be roughly distinguished into two classes: embryonic stem cells (ESCs) and adult/somatic stem cells (ASCs). UCB-derived S/PCs could be catalogued among the latter. In the last ten years ESCs have been messianicly referred to as units of development and of regeneration [48]. Despite being ethically controversial [49], ESCs contained in the blastocyst were once considered as the sole reserve of pluripotent stem cells [50], while ASCs were until recently retained to be lineage/ tissue-restricted. However recent papers have started manifesting deception towards the ESC research endeavour [51]. Indeed, although the use of ESCs has advantages over ASCs, viz a higher autoreplicative capacity, the maintenance of pluripotentiality and absence of age-effects, on the other hand it poses problems related to an increased likelihood of tumor formation after in vivo transplantation (e.g. teratocarcinomas) along with the possibility of an improper differentiation, due to epigenetic culture instability [51]. Recent evidences of ASC plasticity have widened the horizons. Indeed, UCB-derived S/PCs have been proven to give rise to several non-hematopoietic cells [52], such as neural, bone and muscle forming cells [53] or endothelial cells [54,55]. Autologous bone marrow transplantation has been successfully experimented in the treatment of patients with severe heart failure [56], suggestive of a future auspicious

Table 2 – UCB collection, biological qualification and storage [22]. Collection methods: no relevant advantage of the one method over the other [23–27]

Screening for biological qualification

Cryopreservation

In utero: prior to placental expulsion; gives better volumes (42–240 mL, with an average of 103 ± 49 ml) at a higher risk of contamination Ex utero: after placental expulsion; compromises volumes, lower risk of contamination

Contamination and infectious transmissible diseases (HIV, hepatitis, etc.)

Storage at −196 °C in liquid nitrogen, as firstly experienced by Koike in 1983 [28]

Public versus Private banking [28]

Public: does not cost the mother or her insurance provider; for altruistic purposes only. Private: for autologous purposes; expensive; ethical concern (for example private banking is legal in the US but it is not legal in Italy, owing to the fact that there is lack of convincing evidences on the efficacy of autologous donation and loss of valuable units for allogeneic purposes.

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application of UCB-derived S/PCs. Out of those, UCB mesenchymal stem cells (MSCs) will endow clinicians with a new potential tool in cellular therapeutics [57–59], though further studies are still required. On the other hand, the small ratio of the collected UCBvolume/single donor, and the subsequent paucity of overall cells per sample, claim for the development of experimental protocols in order to induce proliferation of UCB-derived S/PCs, to the end of obtaining a suitable cell dose for an eventual tissue-repairing therapy [60]. The whole process should be under strict control in order to prevent anomalous proliferations of the engrafted cells and tumorogenic effects (e.g. increased telomere length [61]) also in long term surviving patients after the treatment. In order to induce proliferation, S/PCs should be exposed to a growth factor enriched environment [52,62]. A wealth of data and a flourishing literature around alternative protocols have made the UCB-derived S/PC culturing a hot issue in recent years [63–67]. Therefore, either for transplantation or cellular therapeutic purposes, the elucidation of the mechanisms governing self-renewal and differentiation is needed to properly drive the in vitro expansion. Results from pilot clinical trials of transplants using expanded UCB-HSCs have shown no adverse effects in the patients. However, further clinical trials must be conducted in order to guarantee the safety of these expanded cells [66]. In the regenerative medicine endeavour, literature refers to UCB-derived S/PCs as an ethically valuable alternative to the use of ESCs, whose derivation has been extremely questioned during the last few years. Indeed the modalities of UCB collection, along with the relative ease of the almost costless procedure, overpass the moral obstacle withstanding large-scale ESC studies and clinical trials [49]. When appropriate studies will shed light on the mechanisms underlying the differentiation of UCB S/PCs into non-hematopoietic cells, UCB could perhaps represent the principal source of S/PCs for cellular treatments.

2.

UCB and proteomics

2.1.

Why a proteomic approach

Until recently, previous approaches to biomarker identification have sought to find single molecules indicative of normal or anomalous conditions – in order to distinguish healthy cells from diseased ones – progenitors or terminally-committed mature cells. Indeed, the molecular complexity is an emerging property of living cells and reductionist approaches have so far restrained an exhaustive understanding of the molecular processes. The proteome is a cell-specific protein complement to the genome and encompasses all the proteins which are expressed in a cell at the given time and conditions (normal, stress, disease, tumor) in which the experiment is performed [68–70]. The proteomic approach [71,72] could reveal a useful, but not the unique [70], tool to gain insight of the cellular complexity. UCB-derived S/PCs could potentially differentiate into several cell lineages [53–57]. Recently, it has been eagerly discussed whether the highly-plastic potential of HSCs and, in particular, of MSCs might allow these cells to transdifferentiate, under appropriate conditions, into multiple non-hematopoietic cell types (brain, liver, kidney, lung, skin, gastrointestinal tract,

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skeleton muscle and cardiac myocyte) [73,74]. Unfortunately, despite early successes, UCB-S/PC application has been so far limited to a narrow range of experimental treatments [32], especially as an alternative and richer source of HSCs than bone marrow for transplantation purposes [75]. More than 10,000 UCB transplants have been performed so far [76], but the molecular events directing self-renewal and multilineage differentiation events are still poorly understood, though some cytokines have been identified as crucial triggers (interleukins: IL-1, IL-3, IL-6; thrombopoietin, granulocyte-macrophage colony stimulating factor — GM-CSF and stem cell factor — SCF; fibroblast growth factors such as FGF-4) [60]. Besides, proteomic analyses of UCB samples from different donors could lead to the detection of specific markers for quality control of UCB units prior to and after cryostorage. It is therefore futile to further stress the reason why, in recent years, the greatest scientific efforts have been made towards this direction. To gain a comprehensive understanding of HSCs from UCB, systematic proteomic surveys have been performed [77–80] (Fig. 2). Since quantitative proteomics results are extremely accurate in proteomics [81], sample collection and preparation time and methods should be highly standardized in order to eliminate, or at least reduce, unwanted human errors or ab initio heterogeneity. Adequate proteomic surveys should address UCB-derived cell populations at the same stage of differentiation. Hence, the first studies were targeted to the definition of the molecular markers expressed by the UCB S/PCs at different stages of commitment. UCB S/PCs should be isolated either to induce proliferation [60] or for preliminary enrichment before the experimental phase [82]. In the latter case, MACS or FACS are usually used [82]. Magnetically-labeled of fluorescent-isothiocyanate (FITC)labeled antibodies for this purpose are mainly aimed at recognizing a handful of membrane glycoproteins such as CD133+ [83], CD34+ [84–87], CD45+ [58], each one representing a specific step in the commitment process [58]. Alternative, but currently poorly characterized Lin− progenitor cells lack lineagespecific markers [86]. Multiple separations can be simultaneously performed by means of multiple wavelength fluorophore-labeled antibodies [88]. FACS [88] and MACS [86] in the field of stem cell biology have become an indispensable tool for defining and separating rare cell populations with a high degree of purity. Steady progress has been made in this regard, but the intrinsic lability of the stem cell phenotype presents a different challenge. Indeed, there are many technical caveats, such as untoward triggering of intra-cellular cascades by antibody binding to the targeted membrane molecules, which may induce partial activation/maturation processes in the isolated populations and have pitfalls on their proteome variability [87]. Just to give an example, an anti-CD34 antibody has been shown to induce tyrosine phosphorylation in bone marrow-derived CD34+ cells [89]. Nonetheless, this is not true for every isolated subset population. For example, Lin− cells are selected through negative depletion. Thus, neither antibody binding nor activation of signaling pathways is expected in this case [86]. However, these relevant considerations have not so far hampered diffusion of FACS [89] and MACS [86] as the technologies of choice for reporting and characterizing rare cell populations

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Fig. 2 – Umbilical cord blood (UCB) has been so far analyzed with several proteomic approaches. UCB units are collected (1) and tested for several diseases (2). UCB from healthy donors are stored in liquid nitrogen (3) under appropriate conditions. Thawing protocols are applied in order to isolate UCB stem cells from the mononuclear cell fraction after Ficoll–Paque centrifugation (4). The targeted population among UCB stem cells is isolated with Magnetic activated Cell Sorter (MACS) methods (5) and purity assessed by FACS (6). Isolated populations could be cultured in vitro (long term culture-initiating cell — LTC-IC, cobblestone area forming cell — CAFC) or in vivo (engrafted in non-obese diabetic/severe combined immunodeficient — NOD/SCID mice) prior to transplantation (5). This step has been rarely performed during proteomic experiments [99]. Cells are gently sonicated (8) (sometimes on ice in order to prevent precipitation of low-abundant protein or denaturation of proteins from cell pellets [79,80]. A step of centrifugation is scheduled (9): supernatants and pellets usually undego a separated analysis [77]. 2D-GE (often IEF + SDS-PAGE [80]) operates an important separation of proteins (10a). Spots of interest are manually [79] or robotically [100] cut from the gel (11a) and digested overnight (generally with trypsin) before the final MALDI-TOF (12a) step. A parallel approach is made up of a 2D-Strong Cation Exchange (SCX) + rp-HPLC) (10b) which is followed by nano-LC-ESI-MS analysis (11b). In-gel digested spot (also from 1D SDS-PAGE) could be simultaneously or sequentially investigated with LC-ESI-MS (or tandem MS) methods (11a ➔ 11b). MS/MS fingerprinting for a more precise identification of the peptides could be performed as well. Indeed, the last step consists of the interrogation of informatics archives such as MASCOT (13): queries that show high matching probability with experimental data are catalogued as final results.

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such as stem cells [89], although further studies on the effect of the interaction between stem cells and the antibodies used for their selection, as well as the possible impact of this contact on HSC quality are awaited. Both separation methods could be used in sequence: the first MACS step to isolate the desired fraction, the FACS step to verify its purity [79] (higher score values range from 90% to 98% depending on the specificity of the marker).

2.2.

CD34+: a suitable marker for targeted proteomics

The CD34 antigen is a highly glycosylated type I transmembrane protein with a molecular weight of 105–120 kD [90]. Detailed structural analyses and cloning studies have confirmed that CD34 is a sialomucin, whose carbohydrate moieties in the N-terminal region have a regulatory activity [91]. It can be phosphorylated by a variety of kinases, including protein kinase C (PKC) and tyrosine kinases [90]. More than 20 monoclonal antibodies are available for different CD34 epitopes, at present [92]. CD34 is not a universal marker of UCB stem cells [93]. However, since the UCB-HSC fraction still represents the most clinically relevant one to date, it is natural that the first proteomic studies have been performed on this population. Indeed, CD34 has been used as a convenient marker for human hematopoietic S/PCs for autologous and allogeneic transplantation, resulting in a rapid reconstitution of all hematopoietic lineages [94]. Most colony-forming cells are found in the CD34+ fraction [95,96].

Table 3 – Relevant UCB-HSC proteomic studies. Authors

Year

Reference

Methods

Zenzmaier et al Tao et al Zenzmaier et al Liu et al

2003

[79]

2D-SCX-rp-HPLC, ESI-MS/MS;

2004 2005

[100] [80]

2006

[77]

2D-IEF-SDS-PAGE, MALDI-TOF; 2D-IEF-SDS-PAGE, nano-LC, ESI-MS/MS; 2D-IEF-SDS-PAGE, MALDI-TOF, 1D-GE, 2D-SCX-rp-HPLC, nano-LC, ESI-MS/MS.

UCB displays a lower number of CD34+ cells than bone marrow, though being richer in colony-forming cells [97–99]. The sialomucin CD34 is also expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma [100], thus reinforcing the idea of CD34+ being a specific marker for proliferating cells. Proteomic techniques have been used to investigate CD34+ populations from UCB [77,79,80,100,101] (Fig. 3, Table 3). Thorough analyses of UCB-derived CD34+ cell populations have been performed by Zenzmaier and his group [79,80]. The declared intent of the 2003 study [79] was to identify key factors that govern ex vivo expansion of HSCs for a safe and sufficient propagation of individual cord blood. UCB samples obtained from full-term deliveries, after informed consent of the mother, were layered on Ficoll–Paque density gradient and centrifuged at 1150 g for 20 min. Since the proteome of a cell is a dynamic

Fig. 3 – Graphics show data from proteomic analyses of CD34+ fractions from UCB. Vesicular transporters and heat shock proteins have been listed as cytoskeletal and other proteins respectively by the authors [77,79,80,10077,79,80,100]. Gel-based separation methods followed by in-gel digestion and MS analysis yielded a lower number of unambiguously identified proteins when used alone [80,100]. Intrinsic limitations affecting gel-based methods (scarce sensitivity towards low-abundant and low-molecular weight proteins) were stressed by the heterogeneity of the samples and should affect the eliciting criteria for the evaluation and choice of the most suitable approach in future studies. It is inevitable to note that preliminary data from gel-based or non-gel-based separation methods displayed a wider variety of potential proteins, which turned out to be highly redundant when ultimately identified with MS. Post-translational modifications are likely to concur to this phenomenon.

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parameter, when pooling heterogeneous samples together Zenzmaier et al. [79] were aware of the influence that this approach would have had on their results, but they also believed that this was the quickest method to circumvent the low-cell dose obstacle in their experiment [79]. The mononuclear cell interface was collected and the CD34+ fraction was extracted and enriched with immunomagnetic methods, while FACS was used to definitely assess the purity of the fraction. This phase is crucial, since a small amount of available cells would negatively affect the outcome of the proteomic analysis, especially as it regards low-abundant proteins, whose signals are masked by the most frequent ones [81]. A preliminary attempt to decipher the UCB-HSC proteome was put forward by applying 2D-HPLC to a crude lysate of HSCs and by trying to identify as many proteins as possible by ESI-MS/ MS. The output dataset allowed for the first time an identification of a limited number of CD34+-cell-specific proteins. 2D-HPLC consisted of a combination of ion exchange chromatography followed by rp-HPLC. 2D-HPLC is a valid alternative to 2D-GE [102], in particular when a large number of proteins/peptides are under simultaneous investigation [103]. Eluted peptides were introduced into the ESI-MS and ionized to be further transferred online to a heated capillary of an IT-MS. The most intensive ions underwent MS/MS analysis and the output data compared against Mascot program [104] and Fasta database [105]. Only 215 proteins were isolated, 31 of which were membrane proteins, 28 cytoskeletal, 53 nuclear, 4 signaling, 7 extracellular, 49 catalogued as “other” proteins, and 43 enzymes [79]. Obviously, the small number of the individuated proteins did not reflect the whole proteomic network within the UCB-HSCs, but at the same time it represented a breakthrough in the complexity of these cells and a first draft of the protein fingerprint of healthy UCBHSCs. After this preliminary phase, Zenzmaier and his group focused their attention on individuating cell markers of UCB-HSCs, which could be likely used for quality control analysis of UCB during storage. Zenzmaier and co-workers performed a preliminary 2D-GE [106], followed by in-gel digestion and nano-LCMS/MS for protein identification [80]. 5 UCB samples were treated with the same preparation protocol as in Liu et al. [77], whereas the addressed UCB-HSC population was CD34+, CD45+. First-dimension IEF consisted of a pH gradient of 3–10 or 4–7 along 7 cm strips, while second-dimension SDS-PAGE was performed after reduction of disulfide bonds and alkylation of cystein residues by means of dithiothreitol (DTT) and iodacetamide respectively. Silver stain was used to reveal the protein spots on the gels. By means of a clean very sharp scalpel, 52 spots of interest were excised from the gel and digested overnight before being further analyzed with a nano-HPLC online with a ESI-MS/MS. High molecular-mass proteins were not found when interrogating search engines [107], probably due to significant proteolytic digestion. Out of the 52 digested spots, 22 proteins were reliably identified [80], while only 3 proteins matched with the previous 2D-HPLC study of the same group [79]. Particular attention was attracted by the heat shock protein 27 (hsp27) since it is involved in high protein expression as well as protein turn-over. Cytoprotective and oncogenic functions have been also attributed to hsp27 [108]. Its presence has been interpreted as a clear signal of proteomic “immaturity” of stem cells [80]. Western blot analysis clearly showed that hsp27 was present in

the CD34+ fraction and, in general, in the mononuclear cell fraction from which UCB cells had been collected after Ficoll– Paque identification [80]. On the other hand, hsp27 expression was down-regulated during differentiation into leukocytes (CD45+/CD34−/AC133−). Another proteomic survey on UCB CD34+ fraction intended to investigate how the protein expression varied at different maturation stages within the hematopoietic hierarchy [100]. CD34+ was selected as the “immature” fraction, while CD15+ cells were chosen as the mature myeloid-differentiated population [100]. In fact, CD15 (Lewis-X or X-apten) is expressed mainly in mature granulocyte (neutrophils and eosinophils) and to a varying degree on monocytes, but not on lymphocytes or basophils in peripheral blood [109–111]. Once again [79,80], the CD34+ fraction was immunomagnetically recovered from the low-density mononuclear cell layer after Ficoll–Paque gradient of heparinized UCB within 12 h from collection. CD34+ cell-depleted populations were cultured overnight in the presence of specific growth factors (SCF, GM-CSF) and nutrients (L-glutamine) and fetal bovine serum. Subsequently, the CD15+ fraction was isolated with the immunomagnetic bead methods. FACS analysis assessed the purity of cell fractions. CD34+ fraction was furtherly cultured for 5 days. Proteomic analysis consisted of a 2D-GE (IEF, MOPS-buffer PAGE) and MALDI-TOF. Overall 460 protein spots were detected on each gel from both populations. 112 and 15 protein spots were found to be differentiallyexpressed or post-translationally modified in CD34+ and CD15+ cells respectively. This suggested that CD34+ cells have a relatively larger proteome than mature CD15+ myeloid cells and production of many S/PC-associated proteins ceases or is progressively down-regulated as the CD34+ cells undergo differentiation. A MALDI-TOF analysis of the differentiallyexpressed spots lead to the final identification of 47 proteins which could be classified into a variety of functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. A broad array of proteins were found to be selectively expressed by the CD34+ population, whose global proteome appeared to be more consistent. The expression of many stem-cell associated proteins totally ceased or dramatically decreased while CD34+ cells underwent myeloid maturation. It is more than a coincidence that various heat shock proteins and chaperones were found to be predominantly present in CD34+ “immature” cells, along with proteins important for vesicular transport. These data confirm the results from Zenzmaier et al. [79,80]. Protein folding and quality control systems in the endoplasmic reticulum and in the secretory pathway parallely function as part of post-translational checkpoints as to ensure the fidelity and regulation of eukaryotic protein expression [112–114]. From these studies [79,80,100] a peculiar role emerges for regulatory proteins in the earlier stages of cellular maturation, when their expression seems to reach its climax. In addition, more than a dozen proteins were found to be expressed only by the CD34+ fraction. Among those proteins, the prostatic binding protein (PBP or RKIP) was identified. Two roles have been proposed for this protein in accordance with literature: the first as inhibitor of the MAPK by preventing its phosphorylation, the second as a mediator of G protein coupled receptor desensitization by working as a decoy for G protein-dependent receptor kinase 2 (GRK2). Both activities

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would justify a predominant role for PBP in taking part in proliferation and homing regulatory events [115–117]. The latest paper on UCB-HSC proteomics is perhaps the most complete one [77]. CD34+ cells were isolated by MACS from mononuclear cell fraction of Ficoll–Paque centrifuged UCB after 24 h from collection and FACS assessed their purity. 2D-GE, in-gel digestion of selected spots and MALDI-TOF analysis of cell lysates concluded the first phase of proteomic assays. The second phase of experiments was characterized by rp-nano HPLC which followed both strong cation exchange liquid chromatography (SCX-LC) and in-gel tryptic digestion after 1D-GE of the supernatant and insoluble pellet, respectively. Rp-HPLC was online with an ESI-MS/MS analyzer. In parallel, Liu et al. performed semiquantitative RT-PCRs in order to evaluate the levels of transcriptional expression of some genes of interest. Semiquantitative RT-PCR results were compared with the data from other transcriptomic papers on CD34+ populations, which included expressed sequence tag (EST) sequencing [118–120], serial analysis of gene expression (SAGE) [121], and cDNA or oligonucleotide microarray [122–127]. Silver staining of the 2D-GE positively evidenced 478 protein spots. Out of these, 436 had a low-molecular weight (below 40 kDa) and 431 were acidic proteins with distributions limited to pI values below 7.0. MALDI-TOF analysis revealed 144 unique proteins in this phase. In the second phase of experiments (SCX, 1D-GE, rp-nano HPLC, ESI-MS/MS) 252 proteins were identified. 155 of those 252 proteins had a molecular weight higher than 40 kDa and a pI higher than 7.0. Noteworthy, the authors noticed a low overlap between data from the first and the second phases of experiments. These observations reinforced the idea that the two protocols adopted by Liu et al. [77] are complementary (Fig. 4). In the end, overall 370 unique proteins were identified with the different proteomic methods, some having roles in the cell cycle, chromatin regulation, RNA processes, as well as protein synthesis, folding/modification and degradation [77]. It is noteworthy that c-kit, a hallmark of normal and malignant human hematopoiesis [128], was undoubtedly individuated. In coherence with previous observations, CD34+ cells showed an extremely rich spectrum of expressed proteins, which included characteristic markers of more mature cells from different lineages, such as platelet transmembrane glycoprotein IIb [129] and heavy, kappa and lambda chains of immunoglobulins, that are specific for B lymphocytes. More intriguingly, several nerve, gonad and eye-associated biomarkers (Pax-6, tropomyosin, glial maturation factor and so on) were evidenced by the proteomic analysis, in accordance with in vitro induced differentiation of UCB stem cells into non-hematopoietic cells [38–41] and previous experiments on CD34+ cell lines [130,131]. It could be proposed that the profile of “immature” cells undergoes a progressive sharpening which limits the overall protein expression in exchange for an increased specificity. However, further studies are indicated. The datasets from mRNA transcripts and proteins seldom matched (approximately 40% of the cases), in accordance with previous results from analogue surveys [132,133]. In particular, mRNAs which could correspond to the already identified proteins were often undetectable. Whether this could be attributed to an actual shortcoming of the adopted protocols

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Fig. 4 – Graphics show data from proteomic analyses of CD34+ fractions from UCB by means of two different approaches: 2D-HPLC followed by ESI-MS analysis, 2D-GE and MALDI-TOF. In row A we compare the outcome of those approaches from two different papers by Zenzmaier et al. [79,80], while in row B we refer to the data from Liu et al. [77]. Notably, there is a very low overlap percentage (approximately 4% of the overall proteins). Taken together, these results seem to validate the statements from the aforementioned authors [77,79,80] on the complementarity of those techniques. High discrepancy could be underlined between [80] and [77] when focusing on 2D-GE + MALDI — TOF results. This is mainly due by the fact that in the first study [80] UCB stem cells from 5 donors where separately analyzed and common spots after 2D-GE (52 spots) were furtherly investigated. On the other hand, in [77] UCB units from 20 donors were pooled together, a unique analysis was performed and 226 protein recognized. Notwithstanding this, rare proteins could be even more diluted by the pooling step. However, it is yet to be elucidated whether the numerical improvement of data framework from heterogeneous samples ends up to affect experimental reliability and reproducibility.

or to an intrinsic characteristic of cellular systems is yet to be demonstrated. What is already known for certain is that posttranscriptional control strongly contributes to the myeloid maturation program of hematopoietic S/PCs from different sources (bone marrow, peripheral blood or UCB) [134–137]. Also post-translational modifications are regulatory events of extreme importance in multiple stem cell populations [121] and, perhaps in UCB-HSCs too, as Liu et al. [77] suggested when justifying the great redundancy of proteins individuated from 2D-GE spots. Whilst big strides are being made in UCB-HSC proteomics and transcriptomics, protein acetylations and phosphorylations have been hitherto almost ignored [138,139]. Future research projects will have to fill this gap. Furthermore, the biosyntheses of 15 proteins identified in the study by Liu et al. [77] appeared not to be completely interrupted in spite of the fact that corresponding antisense RNAs were found either in

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the transcriptome data, either in literature. Nevertheless, antisense mRNA could represent another fundamental checkpoint in the modulation of protein expression, in like fashion of microRNAs studies on hematopoietic lineage differentiation [140]. Recent achievements in mass spectrometry-based quantification methods, such as extracted ion current (XIC), ICAT or iTRAQ could complement existing knowledge in prospective studies [141]. The analysis and description of the processes that regulate HSC development and the functional activity of mature cells has been improved with proteomic approaches for the characterization of proteins, their interactions, and their relative quantification; moreover, proteomic techniques will probably facilitate comprehensive screening of a large subset of marker proteins, thus allowing evaluation of the integrity, functionality and proliferative capacity of HSCs prior to transplantation [138,139,142].

2.3. MSCs and cellular therapeutics: recent proteomics advances Although UCB has actually found a proper collocation only in the clinical transplantation endeavour, recent discoveries about UCB-derived S/PC plasticity have alimented both debate and investigations on the potential of these cells for cellular therapeutic treatments [52–56]. In this view, MSCs likely represent a valuable resource [57–59], since they are multipotent progenitors which are able to self-renew and terminally differentiate into multiple lineages, including bone [143,144], cartilage [145], tendon [146], adipoid [147], neural [148], hematopoietic [149] and stroma tissue cells [150]. Moreover, they can be easily obtained from bone marrow or other sources and cultivated, though not being ethically stigmatized [151], and could be used to sustain HSC expansion in co-cultivations [39,150]. In particular, MSCs could be suitably isolated from UCB, although no or few markers have been deemed as MSC-specific, thereby resulting in a general ambiguity regarding the makeup of the MSC population [143,144]. Early, albeit fundamental, investigations on UCB-MSC proteome have been recently performed [151–159] (Table 4) and reviewed [160]. Thus we will only briefly introduce them. At first, proteomic approaches to UCB-MSCs were focused on drawing a preliminary map of proteins. One of these early studies lead to the detection of approximately 2037 spots with 2D-GE and the final MALDI-TOF/TOF identification of 205 Table 4 – Relevant MSC proteomic studies. Authors

Year

Reference

Methods

Wang et al. Feldmann et al. a Salasznyk et al. Kratchmarova et al. Jeong et al. a Lee et al. Zhang et al. Roubelakis et al. Kim et al. a

2004 2005 2005 2005 2006 2006 2006 2007 2008

[151] [152] [153] [154] [155] [156] [157] [158] [159]

2D–GE, Q–TOF MS; 2D–GE, MALDI–TOF; 2D–GE, 2D–HPLC, MS; MS approaches; 2D–GE, MALDI–TOF; 2D–GE, MALDI–TOF; 2D–GE, MALDI–TOF; 2D–GE, MALDI–TOF 2D–GE, ESI Q–TOF, Western blot.

a

MSCs from UCB sources.

proteins, among which 145 different proteins and isoforms or PTMs [152]. The identified proteins could be grouped into several functional categories, such as metabolism, folding, cytoskeleton, transcription, signal transduction, protein degradation, detoxification, vesicle/protein transport, cell cycle regulation, apoptosis, and calcium homeostasis [152]. In 2006, Jeong et al. performed a 2D-GE and MALDI-TOF analysis, which yielded identification of 32 proteins (most of which were chaperones) out of 35 protein spots of interest [155]. Proteomics (2D-GE and MALDI-TOF) has been also used to determine specific markers during differentiation and maturation processes, such as commitment towards adipocytes [156] and osteoblasts [157,159]. Thirty-two protein spots were shown to have different expression levels between MSCs and adipocyte derived from these cells. Among these, eight proteins were identified by MALDI-TOF/MS, as the following: syntaxin binding protein 3, OSBP-related protein 3, phosphodiesterase, glycophorin, immunoglobulin kappa chain variable region, peroxisome proliferative activated receptor gamma (PPAR-gamma), bA528A10.3.1 (novel protein similar to KIAA01616, isoform 1), and T cell receptor V-beta 4. Four proteins in particular were associated with adipogenesis: syntaxin-3, OSBP-related protein 3, PPAR-gamma and glycophorin [156]. During osteoblast differentiation of MSCs [157], 2D-GE revealed 102 spots with at least 2.0-fold changes in expression. 52 differently expressed proteins were successfully identified by MALDI-TOF-MS [157]. These proteins were classified into 7 functional categories: metabolism, signal transduction, transcription, calcium-binding protein, protein degradation, protein folding and others. An analogue investigation has been recently performed [159] by means of 2D-GE, ESI Q-TOF and Western blot. Eleven proteins were found to be differentially-expressed by MSC-derived osteoblasts when compared to undifferentiated MSCs. Out of these proteins, 4 were confirmed to be associated with osteogenesis: PGAM1, VBP1, hsp27 and β-actin. These proteins were down-regulated during differentiation events [159], in a likewise fashion to CD34+ myeloid differentiation, when maturation paralleled a diminished expression of heat shock proteins [100]. Another interesting approach to the MSC proteome has been performed by other groups [151,154]. MSCs could be induced towards specific lineage commitment through exposure to a growth factor enriched environment. In the cited studies, the authors relied on peculiar growth factors, such as transforming growth factor β 1 [151] and/or epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) [154]. Proteomics analyses have been conducted both on un-induced MSCs and MSCs exposed to growth factors, in order to determine the molecular changes upon induction of commitment towards a specific lineage at the protein scale. As a result, Wang et al. [151], as well as Kratchmarova et al. [154] concluded that the proteomic profile closely paralleled phenotypic changes towards smoothmuscle [151] or bone [154] differentiation of MSCs. On the other hand, Kratchmarova et al. [154] also noticed that closely-related growth factors such as EGF and PDGF did not induce the same kind of differentiation events, since only the former was proved to be likely responsible of osteoblast differentiation of MSCs [154]. Thus, closely-related signals finely tune different and specific differentiation pathways.

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These preliminary studies likely constitute an early inventory of proteins expressed by immature MSCs and MSCs differentiating towards specific lineages. These data will hopefully ease a final application of (UCB-derived) MSCs for cellular therapeutic treatments.

3.

Conclusions

A long series of reasons have recently led to consider UCB as a valuable and alternative source of stem cells [3,161] such as, above all, the high degree of plasticity of UBC-derived S/PCs. Indeed, in vitro UCB S/PCs could be induced to maturate into neural, bone, muscle and endothelial progenitors [53–57,143–150], though the mechanisms underlying these processes have not yet been elucidated. These results have opened new prospective scenarios in the field of cell therapy and regenerative medicine and allowed to compare UCB-derived S/PCs to ESCs, whose ethically-questionable derivation has been provoking significant debates [49]. UCB-derived S/PCs which have already found a clinical application belong to the HSC fraction. UCB-HSCs are now currently used as a valuable resource in the transplantation endeavour in alternative to bone marrow and peripheral blood [19]. In particular, CD34+ populations represent the engine of the engraftment process [94–99]. In order to unravel their full potential, several proteomic approaches have been recently gaining momentum. The depths of the molecular mechanisms which mould the complexity of CD34+ populations have been fathomed by means of electrophoresis, chromatography and mass spectrometry [77,79,80,100,101]. The proteome of UCBderived CD34+ cells was richer than the one of more mature cells (CD15+ [100]) and nerve, gonad and eye-associated proteins were unambiguously observed in the immature fraction [77,130,131]. From these results it could be deduced that the commitment process may represent a bottleneck to protein expression, whereas the acquisition of a mature phenotype parallels with an overall reduction of protein variability. Proteomics results have been compared to transcriptomic data [100] from other surveys [118–127]. Transcriptomic and proteomic data rarely matched [77,132]. Post-transcriptional and post-translational regulatory events could be responsible for this misalignment. However, in addition to various biological factors, it should be considered that the poor correlation between transcriptomic and proteomic data could be quite possibly due to the inadequacy of available statistical tools to compensate for biases in the data collection methodologies [133] as well as to the different bioinformatic approaches employed to analyze and integrate data from either proteomics or genomics studies. The central core of individuated proteins was constituted by heat shock proteins, chaperones and vesicular transport proteins [79,80,100], likely suggestive of a cell effort to protect yet-existing proteins rather than synthesizing new ones. Indeed, the proteome of more immature HSCs appears to be geared to protect from oxidative damage, as it had already been suggested by Unwin et al. [132]. The great majority of the individuated spots with 2D-GE methods were redundant. It is evident that post-translational modifications, such as phosphorylations and acetylations

477

should be investigated as well, since they strongly contribute to the modulation of the proteome activity. Furthermore, protein–protein interactions may alter the outcomes of present studies and mask low-abundant or low-molecular weight proteins. Future studies are mandatory in order to increase knowledge on these crucial events, likewise recent transcriptomic studies have delved deeper into the complexity of mRNA transcripts of CD34+ cell populations [162]. In this recent transcriptomic study more than 459,000 transcript signatures have been collected from bone marrow CD34+ cell populations. Hereby, the authors performed an extensive annotation of a large set of CD34+ transcript sequences. This study has finally provided a current view on gene expression in human CD34+ cells and revealed that early hematopoiesis is an orchestrated process with the involvement of over half of the human genes distributed in various functions [162]. Yet the trodden path has delivered a wide range of proteins, but new approaches such as ICAT and iTRAQ should be implemented along the way as to obtain accurate (further [132]) quantitative data as well. Another relevant issue concerns the few data that we actually have on membrane proteomics for the immature and differentiated UCB-derived stem cells. The methods used so far do not allow efficient resolution of the more hydrophobic protein fractions, with a consequent inevitable loss of transmembrane proteins from the resulting proteomic analysis. 1D-GE in combination with LC-MS/MS methods has been adopted [77] with good but not exceptional results. A brilliant solution to this problem has been proposed by Nunomura et al. [141] for mouse ESCs. Their method is characterized by a combination of cell surface labeling with sulfo-NHS-LC-biotin, membrane fractionation, isolation of peptides with immobilized avidin and largescale 2D LC-MS/MS analysis of the resulting biotinylated peptides. As it regards 2D-GE, the IEF step could be substituted with a blue-native one as already experimented for other blood components [163]. In order to reduce human influences on the proteomic analyses, protein microarrays could become a powerful tool for proteomic visualizations with a high degree of standardization [164]. Actually, a major criterion in this kind of studies is the reproducibility of the proteome profile. A key factor in this respect is the heterogeneity of the samples which is a challenging issue when operating with stem cell lines [165,166]. Furthermore, different proteomics approaches likely yield different and often complementary results, as it emerged from Zenzmaier et al. [79,80] and Liu et al. [77]. In vitro experiments and preliminary proteomic data prospect a broader clinical application for UCB S/PCs in the future, while in Italy public debate claims for the definition of new institutional interventions in this hot research endeavour [3]. A series of translational studies have been recently performed, which highlight the potential of proteomics investigations as a powerful tool in the quality control of UCB or in the follow-up of UCB-HSC transplantations [167–171]. For example, functional proteomics studies have been recently performed on UCB serum in order to thoroughly test the influence of smoking mothers on the newborn [167]. These observations could hopefully reveal useful to determine whether UCB from smoking mothers' newborns could be qualitatively acceptable. Moreover, proteomics has been

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proven to achieve distinction of patients with GVHD and those with no problems after HSC transplantation, with extreme sensitivity and specificity, by addressing protein changes of different biological fluids such as urine [168,169], saliva [170] and plasma [171]. Given their potential to differentiate into several lineages [143–150], (UCB-derived) MSCs represent a valuable candidate for cellular therapeutic treatments. Early proteomic studies have been performed on these cells, both to create a preliminary inventory of proteins expressed by the immature fractions upon growth factor induction [151,152,154,159] and to identify critic biomarkers of differentiation events [153–157]. Once again [79,100], more immature fractions likely display a broader protein profile, with chaperones [155] and heat shock proteins [159] representing a key resource during early phases of commitment. Even closely-related growth factors end up to drive welldistinct differentiation processes [154]. However, in-depth knowledge of the molecular mechanisms governing these events is still lacking and these first proteomic studies are just the tip of the iceberg. New directions have been proposed with a focus on the creation of worldwide accepted protocols for the reduction of sample heterogeneity and the improvement of data comparability. In order to complement actual data, future projects will have to be addressed on post-translational modification, membrane protein expression and protein–protein interactions.

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