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Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells

Abstract Currently, more than 30 000 allogeneic hematopoietic stem cell (HSC) transplantations have been performed for the treatment of hematological and nonhematological diseases using HSC from umbilical cord blood (CB). However, the wide utilization of CB as a source of HSC is limited by the low number of cells recovered. One strategy to expand ex vivo CB‐HSC is represented by the use of bone marrow mesenchymal stromal cells (BM‐MSCs) as a feeder to enhance HSC proliferation while maintaining HSC stemness. Indeed, BM‐MSCs have been recognized as one of the most relevant players in the HSC niche. Thus, it has been hypothesized that they can support the ex vivo expansion of HSC by mimicking the physiological microenvironment present in the hematopoietic niche. Due to the role of placenta in supporting fetal hematopoiesis, MSC derived from the amniotic membrane (hAMSC) of human term placenta could represent an interesting alternative to BM‐MSC as a feeder layer to enhance the proliferation and maintain HSC stemness. Therefore, in this study we investigated if hAMSC could support the ex vivo expansion of HSC and progenitor cells. The capacity of hAMSCs to support the ex vivo expansion of CB‐HSC was evaluated in comparison to the control condition represented by the CB‐CD34+ cells without a feeder layer. The coculture was performed at two different CD34+:MSC ratios (1:2 and 1:8) in both cell‐to‐cell contact and transwell setting. After 7 days, the cells were collected and analyzed for phenotype and functionality. Our results suggest that hAMSCs represent a valuable alternative to BM‐MSC to support: (a) the ex vivo expansion of CB‐HSC in both contact and transwell systems, (b) the colony forming unit ability, and (c) long‐term culture initiating cells ability. Overall, these findings may contribute to address the unmet need of high HSC content in CB units available for transplantation.


Significance statement
To date, more than 30 000 allogeneic hematopoietic stem cell (HSC) transplantations for the treatment of hematological and nonhematological diseases have been performed using HSC from umbilical cord blood (CB). However, the wide utilization of CB as a source of HSC is limited by the low number of cells recovered and thus they need to be expanded. This article shows that placenta-derived cells represent a valuable option and can support the ex vivo expansion of HSC and hematopoietic progenitor cells. Placenta-derived cells could contribute to address the unmet need of high HSC content in CB units available for transplantation.

| INTRODUCTION
During the last 20 years, umbilical cord blood (CB) has become an attractive source for allogeneic hematopoietic stem cell (HSC) transplantation in addition to other sources such as bone marrow (BM) aspirates. [1][2][3][4] Indeed, CB is easily available with a low risk of graft vs host disease and a high presence of HSCs and hematopoietic progenitor cells (HPCs). However, the limited number of HSC and HPC obtained from a single CB unit remains insufficient for the complete reconstitution of BM hematopoiesis in an adult patient. 1,5 Many efforts have been made to circumvent this limitation by a double cord transplant, 6 or by the ex vivo expansion of the HSC and HPC. 7 9,10 In addition, copper chelating agents 11,12 or transcriptional activators and inhibitors [13][14][15] have also been used to increase HSC and HPC numbers for transplantation.
Currently, the use of feeder cells in combination with various cytokine cocktails to mimic the niche microenvironment is being investigated. 1,7,8,16 Importantly, in the HSC niche, mesenchymal stromal cells (MSCs) play a pivotal role by regulating the complex molecular signaling involved in HSC homeostasis. [17][18][19][20] Nowadays, BMderived MSC (BM-MSC) represent the elective source of MSC considering their function in the HSC niche. However, over the years, several groups reported that MSC derived from perinatal tissues, such as amniotic membrane, 21 chorionic villi, [22][23][24] maternal decidua basalis, 25,26 and from the umbilical cord (including Wharton's jelly), 27,28 can constitute an alternative feeder to BM-MSC.
During fetal development, the placenta partly supports hematopoiesis, and thus it is conceivable that it is endowed with characteristics common to the hematopoietic niche. [29][30][31] Moreover, perinatal tissues are considered biological waste after term delivery and are therefore widely available.
Of particular interest are MSC derived from the amniotic membrane of term placenta (hAMSC). They present a phenotype consistent with MSC from fetal membranes, as suggested by a consensus paper established during the First International Workshop on Placenta-Derived Stem Cells in 2008. 21,32 In fact, these cells are marked by the classical MSC proteins CD90, CD44, CD73, and CD105, while they lack hematopoietic markers CD45, CD34, CD14, 33 and human leukocyte antigen-antigen D-related (HLA-DR) and present the in vitro differentiation potential toward one or more lineages including osteogenic, adipogenic, chondrogenic, and vascular/endothelial. 21,34 hAMSC also have immunomodulatory activity and are proposed as treatments of inflammatory-related disorders. 35,36 Indeed, several papers reported that hAMSC transplantation triggers regeneration in animal models of liver 37 and lung fibrosis, 38 collagen-induced arthritis, inflammatory bowel disease, dextran-induced severe colitis, experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis), 39 traumatic brain injury, 40 and cardiac ischemia. 41 The therapeutic effects were observed in the absence of cell engraftment in the injured tissue suggesting that hAMSC act in a paracrine manner. 35,36 This hypothesis was confirmed by studies using conditioned medium (CM) derived from hAMSC that also demonstrated beneficial effects in preclinical models of lung fibrosis, 42,43 cardiac ischemia, 44 Huntington's disease, 45 and skin wound in diabetic mice. 46 Herein, we investigated if hAMSC could represent a valid alternative to BM-MSC as a feeder layer to support HSC and HPC ex vivo expansion. We assessed the expansion and maintenance of the most primitive long-term HSC (LT-HSC), as well as the expansion of shortterm HSC (ST-HSC) that are known to rapidly reconstitute the HSC niche. We also investigated the expression of adhesion molecules involved in stemness maintenance and homeostasis of HSC. Our findings suggest that hAMSC can be a valid alternative to BM-MSC for the ex vivo expansion of the CB-HSC that could help meet the growing needs of HSC for transplants.

| Ethics statements
Amniotic membrane-derived MSC (hAMSC) (n = 11) isolated from human term placenta and CB mononuclear cells (CB-MNC) (n = 12) were obtained from healthy women after full-term vaginal delivery or caesarean section.
BM-MSC (n = 9) were obtained from BM aspirates from healthy donors.
Samples were processed after informed written consent. hAMSC SUPPORT EX VIVO EXPANSION OF CORD BLOOD HSC 2.2 | MSC isolation from human BM BM-MSCs were isolated by plastic adherence as previously described. 47 After isolation, MNCs were then seeded at a density of 0.5 Â 10 6 cells/cm 2 in low glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine (200 mM L-Glu), and 1% penicillin and streptomycin (P/S, 10.000 U P and 10 mg/ml; all from Merck, St. Louis, Missouri). Every 3 days, nonadherent cells were removed, and fresh complete medium was added. Cells were expanded until 80% confluency and then detached with Trypsin/EDTA solution (0.25%, Merck) and expanded until passage 2 (p2) by plating at a density of 1 Â 10 4 cells/cm 2 in DMEM complete medium at 37 C in 5% CO 2 .
The cells were cryopreserved in liquid nitrogen at the final concentration of 1 Â 10 6 cells/mL in FBS supplemented with 10% dimethyl sulfoxide (DMSO) (Merck) until use.

| MSC isolation from human amniotic membrane
MSCs derived from the human amniotic membrane (hAMSC) were isolated from human term placentas of healthy women. Isolation, phenotype characterization, and cryopreservation were carried out as previously described. 48 Amnion fragments were digested at 37 C for 9 minutes with 2.5 U/mL dispase (VWR, Radnor, Pennsylvania); samples were then transferred to RPMI complete medium composed of RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% P/S, and 1% L-glutamine. Enzymatic digestion continued with the treatment of the amnion fragments with 0.94 mg/mL collagenase and DNase I (both from Roche, Basel, Switzerland) for approximately 2.5 to 3 hours at 37 C. The resulting cell suspensions were centrifuged at 150g. The supernatant was filtered through a 100-μm cell strainer (from BD Falcon, Bedford, Massachusetts) and the cells were collected by centrifugation. Freshly isolated (p0) cells were then cryopreserved in liquid nitrogen at the final concentration of 1 Â 10 6 cells/mL in FBS supplemented with 10% DMSO until their use.   Cocultures were established either in cell-to-cell contact or using transwell chambers (0.4 μm pore, polycarbonate membrane; Corning).

| Analysis of BM-MSC and hAMSC phenotype
To detect the basal effect of the cytokine cocktail on the ex vivo expansion of CD34 + cells, the control condition represented by the ex vivo expansion in absence of a feeder layer was included.
Cocultures and CD34 + cells cultured alone were incubated for 7 days at 37 C in humidified atmosphere and 5% CO 2 . After 7 days, the mixed population composed of CD34 + HSC and MSC, as well as the CD34 + HSC cultured alone representative of the basal condition, was collected and cell proliferation and viability were determined by Trypan blue exclusion assay. Cells were then analyzed for their phenotype.

| Flow cytometry analysis of ex vivo-expanded CB-CD34 + cells
The ex vivo-expanded CD34 + cells as well as time zero (T0) freshly isolated CD34 + cells seeded to perform the coculture were phenotypically analyzed for a panel of antibodies to identify and discriminate primitive CD34 + cells from the most advanced progenitor.

| Colony forming unit (CFU) assay
The clonogenic capacity of CD34 + cells expanded after 7 days of coculture with BM-MSC, hAMSC, or in the control condition was evaluated by short-term CFU assay. Cells for burst-forming units erythroid (BFU-E), colony-forming unite granulocyte-macrophage (CFU-GM), and CFU-granulocyte/macrophage/erythroid/megakariocyte (CFU-GEMM) were seeded in duplicate in methylcellulose MethoCult H4434 classic medium (Stemcell Technologies) in 35 mm petri dishes (Corning). Cells were incubated for fourteen days at 37 C in a humidified atmosphere at 5% CO 2 according to the manufacturer's instructions. After 14 days, BFU-E, CFU-GM, and multilineage colonies (GEMM) were scored in situ.

| hAMSCs expand both short-term and longterm HSC
Different studies have correlated the proliferation rate with the differentiation state of CD34 + cells. [61][62][63] In order to evaluate the ability of the two MSC feeders not only to favor CD34 + ex vivo expansion, but also to preserve their stemness, the expansion of the primitive progenitor subpopulations, named LT-HSC (identified as CD34 + CD38 À CD133 + ) and ST-HSC (identified as CD34 + CD38 + CD133 À ), was evaluated by immunophenotype analysis (Figure 3).    Moreover, a stronger expansion in the number of CFU-GEMM by BM-MSCs, compared with hAMSCs, was also observed in the transwell setting (4 ± 0.55 SD and 3.75 ± 0.84 SD times higher for 1:2 and 1:8 ratios vs 3.75 ± 1.05 and 3.75 ± 0.84 for 1:2 and 1:8 ratios, respectively, with hAMSCs). Furthermore, similar results were obtained also for BFU-E, with a comparable amount was obtained regardless of the feeder layers used ( Figure 5C).
Finally, previous observations obtained from the phenotype analysis of the different progenitor subsets were confirmed. Indeed, a specific commitment induced by the hAMSCs toward the GMP subset was previously observed (Figure 4, lower panel). These findings were also confirmed with this functional assay. Importantly, both feeders at both ratios in the contact setting were able to induce a significant increase in the total number of CFU-GM colo-  Similarly, hAMSCs showed a trend for a higher amount of LTC-IC than CTR, but the increase was not statistically significant. Indeed, at We confirmed the similar immunophenotype of BM-MSCs and hAMSCs. Concerning CD105, a typical MSC marker, we confirmed its reduced expression by hAMSC at low cell culture passages. 49 Furthermore, both BM-MSCs and hAMSCs expressed CD54 (Intercellular Adhesion Molecule 1, ICAM-1), 66,68 a receptor involved in the repopulation and homing of the transplanted HSC. 18 As a matter of fact, CD54 (ICAM-1)-deficient mice (CD54 À/À ) have an impairment of quiescence and repopulation capability of the HSC niche. 69 We also confirm the variable expression of PDGFR-β, also known as CD140b, in hAMSC, as previously reported. 70,71 The expression of CD140b has been shown to correlate with enhanced survival and expansion after transplantation. 72 Finally, we report that hAMSC highly expressed nestin, which is associated with HSC quiescence and maintenance in the BM niche. [73][74][75] Interestingly, although some previous studies showed the importance of cell-to-cell contact for the maintenance of stemness, 64-67 we observed ex vivo expansion also in the transwell setting condition, suggesting that both MSC can release bioactive molecules that can trigger the ex vivo expansion of HSC cells and its progenitors. These findings support the study by Fong and colleagues that reported how CM of Wharton's jelly MSC was able to expand CB-CD34 + cells better than the use of the cells in contact setting. 76 On the other hand,  8) were seeded on irradiated M2-10B4 cells for 6 weeks in MyeloCult H5100™ medium supplemented with 10 À6 M hydrocortisone with half-medium changes once per week. After 6 weeks, the total cell fraction of each culture was harvested and 50 000 cells were seeded in MethoCult H4435 Enriched™ medium to perform the CFU assay. The total number of colonies obtained was counted after 14 days. The graph represents the number of LTC-IC with the different cell culture conditions. Data are represented as mean ± SD (*P ≤ 0.05, **P ≤ .01) from ≥5 independent experiments. BM-MSCs, human bone marrow mesenchymal stromal cells; CTR, control or no feeder layer; hAMSCs, human amniotic mesenchymal stromal cells greatest self-renewal capacity and gives rise to all hematopoietic lineages throughout life. ST-HSC presents instead a reduced self-renewal potential generating all the hematopoietic lineages for a short period. 77,78 hAMSC were able to favor the expansion ST-HSC, and a strong increase was observed, in particular, in the transwell system. These findings were also confirmed by FACS analysis on different HSC and HPC subsets, where we did not observe any significant differences in the expansion of the most primitive HPC progenitors (HSC/MPP, LMPP, and MLP), whereas relevant differences were appreciable for the most committed CMP and GMP progenitors. 53,54 The large variability observed in the hAMSC transwell system at the highest ratio (CD34 + -HSC:MSC, 1:8) may be related to the high number of cells used, which could ultimately impact cell viability and consequently also the factors secreted in the transwell setting used.
When we evaluated the hematopoietic clonogenic ability of the ex vivo-expanded CB HPC in the different culture conditions, we observed that both BM-MSC and hAMSC feeders were able to generate the different subtypes of colonies. The strong increase in the CFU-GM subset is in line with what was previously reported, 57,79 although, in contrast to what has been reported by others, we did not observe any significant differences in the amount of BFU-E. 80 Lastly, we studied and compared the capacity of the two feeders to ex vivo-expanded LTC-IC able to produce myeloid progenitors for at least 5 weeks. 58,60 This long-lasting population is relevant because it repopulates the HSC niche upon transplantation. 58,60 Here, we report that both BM-MSC and hAMSC were able to support the maintenance and expansion of primitive LTC-IC. We observed a strong proliferative burst of CD34 + HSC obtained in the presence of hAMSC, while stemness properties were maintained, as confirmed by both the immune phenotype and the clonogenic ability. Some of the differences observed in the phenotype and in the capability of hAMSC and BM-MSC to support the ex vivo expansion of CD34 + can be partly explained by the difference in the cell passage. Indeed, while hAMSCs were used at passage zero, this was not possible for BM-MSCs since the isolation process for these cells requires selection by plastic adherence. 33 Furthermore, although for hAMSC it is possible to obtain a high amount of cells directly from the isolation process, this is not possible for the BM-MSCs that require subsequent steps of in vitro expansion.

| CONCLUSION
The present study provides a detailed comparison of hAMSCs with the gold standard feeder represented by BM-MSCs and reports the capacity of hAMSCs to trigger the ex vivo expansion of CB-HSC, while maintaining their stemness properties as well as the capacity to differentiate, originating the different HPC progenitors. Taken together, the results suggest that the hAMSC feeder could putatively be able to reconstitute the HSC repertoire and highlight the potential application of the hAMSCs as a new alternative feeder to BM-MSCs.

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.